Poplar wood rays are involved in seasonal remodeling of tree physiology

Abstract

Understanding seasonality and longevity is a major challenge in tree biology. In woody species, growth phases and dormancy follow one another consecutively. In the oldest living individuals, the annual cycle may run for more than 1,000 years. So far, however, not much is known about the processes triggering reactivation from dormancy. In this study, we focused on wood rays, which are known to play an important role in tree development. The transition phase from dormancy to flowering in early spring was compared with the phase of active growth in summer. Rays from wood samples of poplar (Populus × canescens) were enriched by laser microdissection, and transcripts were monitored by poplar whole-genome microarrays. The resulting seasonally varying complex expression and metabolite patterns were subjected to pathway analyses. In February, the metabolic pathways related to flower induction were high, indicating that reactivation from dormancy was already taking place at this time of the year. In July, the pathways related to active growth, like lignin biosynthesis, nitrogen assimilation, and defense, were enriched. Based on "marker" genes identified in our pathway analyses, we were able to validate periodical changes in wood samples by quantitative polymerase chain reaction. These studies, and the resulting ray database, provide new insights into the steps underlying the seasonality of poplar trees.

Figure 1.

Seasonal differences in ray contents as indicated by electron microscopy of the cambial and wood formation zone (cross sections). Top row, early spring: ray cells show a very dense cytoplasm and are packed with storage compounds, such as lipids (L), starch (S), and protein bodies (P). Bottom row, summer: ray cells show a prominent vacuole (VA) and present many mitochondria (M). Intercellular exchange is facilitated by plasmodesmata (PD). C, Typical cambial cell; F, (xylem) fiber; R, typical ray cell; V, vessel. Left panels show overview, and right panels show enlarged different sectional planes of the cells depicted in the left panels. Bars = 20 µm.

Figure 2.

LMPC of ray cells from poplar wood. A, First LMPC attempt of rays. From left to right: selected ray, signed ray, remains after ray dissection, catapulted ray. RNA yield and quality from these samples were not adequate for microarray hybridization. B to D, Inverse LMPC. B, Cross section of a poplar twig. C, Dissection of the area between the xylem differentiation zone and the central section. D, Remains after removal of ray-enriched wood. [See online article for color version of this figure.]

Figure 3.

MapMan analysis of the 500 most significantly regulated mapped genes (chosen based on the adjusted P values) from summer versus early-spring samples. These genes were imported into MapMan 3.5.1 and classified accordingly. Presented clusters were restricted to those pathways containing 10 or more genes. Since the group of unassigned genes was also not included, only 193 (65.7%) of the 294 genes up-regulated in summer and 115 (55.8%) of the 206 genes up-regulated in early spring are shown. Negative values indicate clusters with higher induction in early spring; positive values indicate clusters with higher induction in summer.

Figure 4.

Pathway enrichment analysis (competitive test). In total, 101 KEGG pathways have been included in the analysis. All significant pathways (P < 0.05) are represented by the negative decadic logarithm of the enrichment P value (i.e. longer bars represent more significant pathways). A, Up-regulated pathways (induced in summer). TCA, Trichloroacetic acid. B, Down-regulated pathways (induced in early spring).

Figure 5.

Seasonal time course of key transcript abundances from qPCR analyses. All values represent numbers of molecules per 10,000 molecules of actin. Black arrows are as follows: I, first visible signs of leaf senescence; II, completion of leaf abscission in 2006; III, start of (leaf) bud break; IV, end of bud break in 2007 (according to Wildhagen et al., 2010). n = 3 ± sd.

Figure 6.

Timetable of seasonal transcript abundance. The inner ring shows seasons throughout the year with unique events as indicated: I, onset of leaf senescence; II, end of leaf abscission; III, start of (leaf) bud break. The outer rings show activity windows of seasonal expression patterns. Color strengths correspond to expression levels, where the darkest colors indicate peak times of marker transcripts.