Arabidopsis DEFECTIVE KERNEL1 regulates cell wall composition and axial growth in the inflorescence stem.

Dhika Amanda,John F. Golz,Kim L. Johnson,Gwyneth C. Ingram,Roberta Galletti,Antony Bacic,Monika S. Doblin,Colleen P. MacMillan

doi:10.1002/pld3.27

Abstract

Axial growth in plant stems requires a fine balance between elongation and stem mechanical reinforcement to ensure mechanical stability. Strength is provided by the plant cell wall, the deposition of which must be coordinated with cell expansion and elongation to ensure that integrity is maintained during growth. Coordination of these processes is critical and yet poorly understood. The plant‐specific calpain, DEFECTIVE KERNEL1 (DEK1), plays a key role in growth coordination in leaves, yet its role in regulating stem growth has not been addressed. Using plants overexpressing the active CALPAIN domain of DEK1 (CALPAIN OE) and a DEK1 knockdown line (amiRNA‐DEK1), we undertook morphological, biochemical, biophysical, and microscopic analyses of mature inflorescence stems. We identify a novel role for DEK1 in the maintenance of cell wall integrity and coordination of growth during inflorescence stem development. CALPAIN OE plants are significantly reduced in stature and have short, thickened stems, while amiRNA‐DEK1 lines have weakened stems that are unable to stand upright. Microscopic analyses of the stems identify changes in cell size, shape and number, and differences in both primary and secondary cell wall thickness and composition. Taken together, our results suggest that DEK1 influences primary wall growth by indirectly regulating cellulose and pectin deposition. In addition, we observe changes in secondary cell walls that may compensate for altered primary cell wall composition. We propose that DEK1 activity is required for the coordination of stem strengthening with elongation during axial growth.

Highlights

We show that CALPAIN OE lines develop a short and thickened stem compared to wt plants, whereas lines with reduced DEFECTIVE KERNEL1 (DEK1) activity have a mechanically weakened stem resulting in a prostrate stem phenotype
AmiRNA-DEK1 stems was significantly reduced compared to wt (Figure 2; Table 1; Fig. S4a). These results suggest that changes in stem diameter in CALPAIN OE and amiRNA-DEK1 lines are due to changes in cell number and size, most notably in the cortex, interfascicular fiber region (IFR), and pith layers
As the size and shape of cells are largely determined by the structure and composition of the cell walls, we investigated whether the altered cellular phenotypes observed in CALPAIN OE and amiRNADEK1 occur because of changes in cell wall morphology, composition, and/or deposition

Summary

| MATERIALS AND METHODS

Wild type (wt; Arabidopsis thaliana Columbia-0 ecotype), plants constitutively overexpressing CALPAIN (CALPAIN OE) (Johnson et al, 2008), and plants constitutively expressing an artificial microRNA targeting the DEK1 transcript (amiRNA-DEK1) and the dek allele in Columbia-0 (Galletti et al, 2015; Roeder et al, 2012) were grown under short-day conditions with 8-hr light/16-hr dark cycle at 21°C for 12 weeks. Fluorescence immunolocalization experiments were carried out following the protocol from Coimbra et al (2007), and imaging was performed on a Leica SP5 microscope (Leica Microsystems, Germany) using laser beam lines of 405 nm (calcofluor white) and 488 nm (FITC; Alexa Fluor 488). 2.9 | Carbohydrate Analysis of cell walls (a) Approximately 100 mg of material from a 3-cm segment of the stem base from 4-month-old CALPAIN OE, wt, and amiRNA-DEK1 plants was used to prepare an alcohol-insoluble residue (AIR) cell wall preparation as described by Pettolino, Walsh, Fincher, and Bacic (2012). Lignin content of the stem (approximately 20 mg) from a 3-cm segment of the stem base from 4-month-old CALPAIN OE, wt, and amiRNA-DEK1 plants was estimated following the protocol of Theander and Westerlund (1986). Two biological replicates and two technical replicates were used for each line

| RESULTS

Findings

| DISCUSSION