Analysis of secondary growth in the Arabidopsis shoot reveals a positive role of jasmonate signalling in cambium formation

Abstract

After primary growth, most dicotyledonous plants undergo secondary growth. Secondary growth involves an increase in the diameter of shoots and roots through formation of secondary vascular tissue. A hallmark of secondary growth initiation in shoots of dicotyledonous plants is the initiation of meristematic activity between primary vascular bundles, i.e. in the interfascicular regions. This results in establishment of a cylindrical meristem, namely the vascular cambium. Surprisingly, despite its major implications for plant growth and the accumulation of biomass, the molecular regulation of secondary growth is only poorly understood. Here, we combine histological, molecular and genetic approaches to characterize interfascicular cambium initiation in the Arabidopsis thaliana inflorescence shoot. Using genome-wide transcriptional profiling, we show that stress-related and touch-inducible genes are up-regulated in stem regions where secondary growth takes place. Furthermore, we show that the products of COI1, MYC2, JAZ7 and the touch-inducible gene JAZ10, which are components of the JA signalling pathway, are cambium regulators. The positive effect of JA application on cambium activity confirmed a stimulatory role of JA in secondary growth, and suggests that JA signalling triggers cell divisions in this particular context.

Figure 1

Tissue composition within the primary stem.No IC has been established in the interfascicular region. However, the FC in vascular bundles has started to produce secondary vascular tissue, which is visible as radial cell files in the xylem area proximal to the cambium (most prominent in the left-hand bundle). All other tissues are of primary origin. Scale bar = 100 μm.

Figure 3

Quantitative analysis of the longitudinal and lateral extension of the IC and the ICD tissue at various developmental stages.

Figure 2

Histological analysis of the basal stem segment at various developmental stages.(a–c) 2 cm stems sectioned immediately above the rosette (a), and 1 mm (b) and 2 mm (c) above the rosette.(d–f) 5 cm plants analysed immediately above the rosette (d), and at 1.8 mm (e) and 3.7 mm (f) above the rosette. Phloem initiation is indicated by arrows (d).(g–i) Sections of 15 cm stems from immediately above the rosette (g), and from 2 mm (h) and 5.2 mm (i) above the rosette. Phloem initiation is indicated by arrows (g).(j–l) Sections of 30 cm stems taken from immediately above the rosette (j), and from 2.7 mm (k) and 7.1 mm (l) above the rosette.IFs, interfascicular fibres; ICD, interfascicular cambium-derived tissue (including IC); DPP, distal pith parenchyma. Asterisks indicate primary vascular bundles. Scale bar = 100 μm; same magnification throughout.

Figure 4

Stress-related genes are over-represented in the group of genes preferentially expressed at the stem base.(a) Biological function of genes identified as preferentially expressed at the stem base. Percentages add up to more than 100 because genes may belong to more than one functional category.(b) Stress-related genes preferentially expressed at the stem base. Ticks indicate whether genes were classified as touch-inducible according to Lee et al. (2005).

Figure 5

Secondary growth phenotype of JA signalling mutants.(a,b) Comparison of acropetal progression of IC initiation (a) and lateral ICD extension at the stem base (b) between wild-type, jaz10-1, jaz10-2 and jaz7-1 mutants.(c,d) Analysis of cross-sections taken from the base of 30 cm wild-type (c) and jaz10-1 (d) stems. Lateral extension of the ICD is indicated by brackets. Scale bar = 200 μm; same magnification in (c) and (d).(e) Comparison of stem diameter at the base between wild-type, jaz10-1, jaz10-2 and jaz7-1 mutants.(f) Analysis of longitudinal IC extension in 25 cm stems of wild-type, coi1-1 and myc2-3 mutants.

Figure 6

Effect of JA application on stems.(a,b) Comparison of mock- (a) and JA-treated (b) stem sections taken from the base of 30 cm plants. Lateral ICD extension is indicated by brackets. Phloem fibres are indicated by the arrow in (b). Scale bar = 100 μm; same magnification in (a) and (b).(c) Quantitative analysis of lateral ICD extension at the stem base in mock- and JA-treated plants.

Figure 7

Analysis of JAZ10 expression dynamics.(a) Whole-plant overview visualizing JAZ10:GUS reporter gene activity. The inset shows a close-up of a root tip.(b–e) JAZ10:GUS activity at the base of 2 cm (b), 5 cm (c), 15 cm (d) and 30 cm (e) inflorescence stems. Rosette leaves have been removed for clarity. Arrows indicate the stem base. Scale bar = 2 mm; same magnification in (b–e).(f) Cross-section from the base of a 2 cm plant. JAZ10:GUS activity is detected in xylem (arrowheads) and interfascicular regions (arrow). Primary bundles are labelled by asterisks. Scale bar = 100 μm.(g,h) Local inducibility of JAZ10:GUS activity by mechanical stimulation. Weak (g) and strong (h) stimulation led to different JAZ10:GUS activity levels (see main text for details). The site of stimulation is marked by arrows. Scale bar = 2 mm; same magnification in (g) and (h).(i,j) Quantitative RT-PCR results demonstrating the inducibility of JAZ10 (i) and IAA5 (j) by various stimuli. Seedlings were mock-treated, stroked by hand for 30 sec (‘touch’), sprayed with MeJA (50 μm) or sprayed with IAA (20 μm), and harvested after 2 and 4 h. IAA5 (AT1G15570) expression was used as a positive control for IAA treatment.

Figure 8

Analysis of the erf104 mutant and H4 transcript accumulation.(a) Comparison of acropetal progression of IC initiation between wild-type and erf104 mutants.(b,c) H4 transcript accumulation in 2 cm stems at 2.5 mm (b) and the actual stem base (c). Arrowheads indicate H4-positive cells in the FC, arrows point to interfascicular regions. Refer to Figure 3 for an overview of IC and ICD extension at this stage. Asterisks indicate primary vascular bundles. Scale bar = 100 μm; same magnification in (b) and (c).