Transcriptome profiling of radiata pine branches reveals new insights into reaction wood formation with implications in plant gravitropism

Abstract

Background: Formation of compression (CW) and opposite wood (OW) in branches and bent trunks is an adaptive feature of conifer trees in response to various displacement forces, such as gravity, wind, snow and artificial bending. Several previous studies have characterized tracheids, wood and gene transcription in artificially or naturally bent conifer trunks. These studies have provided molecular basis of reaction wood formation in response to bending forces and gravity stimulus. However, little is known about reaction wood formation and gene transcription in conifer branches under gravity stress. In this study SilviScan® technology was used to characterize tracheid and wood traits in radiate pine (Pinus radiata D. Don) branches and genes differentially transcribed in CW and OW were investigated using cDNA microarrays.

Results: CW drastically differed from OW in tracheids and wood traits with increased growth, thicker tracheid walls, larger microfibril angle (MFA), higher density and lower stiffness. However, CW and OW tracheids had similar diameters in either radial or tangential direction. Thus, gravity stress largely influenced wood growth, secondary wall deposition, cellulose microfibril orientation and wood properties, but had little impact on primary wall expansion. Microarray gene transcription revealed about 29% of the xylem transcriptomes were significantly altered in CW and OW sampled in both spring and autumn, providing molecular evidence for the drastic variation in tracheid and wood traits. Genes involved in cell division, cellulose biosynthesis, lignin deposition, and microtubules were mostly up-regulated in CW, conferring its greater growth, thicker tracheid walls, higher density, larger MFA and lower stiffness. However, genes with roles in cell expansion and primary wall formation were differentially transcribed in CW and OW, respectively, implicating their similar diameters of tracheid walls and different tracheid lengths. Interestingly, many genes related to hormone and calcium signalling as well as various environmental stresses were exclusively up-regulated in CW, providing important clues for earlier molecular signatures of reaction wood formation under gravity stimulus.

Conclusions: The first comprehensive investigation of tracheid characteristics, wood properties and gene transcription in branches of a conifer species revealed more accurate and new insights into reaction wood formation in response to gravity stress. The identified differentially transcribed genes with diverse functions conferred or implicated drastic CW and OW variation observed in radiata pine branches. These genes are excellent candidates for further researches on the molecular mechanisms of reaction wood formation with a view to plant gravitropism.

Figure 1

Variation in tracheid characteristics and wood properties between compression (CW) and opposite wood (OW) of branches. Ring width, tracheid wall thickness, radial diameter, tangential diameter, coarseness, specific surface, microfibril angle (MFA), wood density and stiffness (modulus of elasticity, MOE) were measured in six wood strips of radiata pine branches using SilviScan 2. Average ring values of each trait were compared between the lower side (CW) and upper side (OW) of the six branches. Tracheid and wood traits in ring 10 representing developing xylem tissues collected for microarray experiments were also compared between CW and OW. Error bars represent the standard deviation of the mean value of each trait. CW and OW variation is statistically significant (P-values ≤ 0.05) except for the two tracheid diameters.

Figure 2

Transcriptome comparisons between compression (CW) and opposite wood formed in branches. Genes differentially transcribed in CW and OW sampled in spring and autumn were identified using radiata pine cDNA microarrays, respectively. Numbers of preferentially transcribed genes identified from developing xylem sampled in spring (a) and autumn (b) were present. Differentially transcribed genes were further compared between the two seasons. A number of genes showed consistently differential transcription in the two wood tissues across the two seasons (c).

Figure 3

Validation of microarray transcription of selected differentially transcribed genes. A total of seven differentially transcribed genes were selected in the validation using reverse transcriptase-multiplex ligation dependent probe amplification (RT-MLPA). These genes include four genes up-regulated in CW: cellulose synthase 3 (PrCesA3), PrCesA11, cinnamic acid 4-hydroxylase (C4H) and plastocyanin-like (PCL); three genes more highly transcribed in OW: peroxidase (PER), E3 ubiquitin protein ligase (UPL1) and retinoblastoma-like protein (RBL). Developing xylem (CW and OW) sampled in autumn for the microarray experiment was used in the validation, including three biological and four technical replicates. Mean log-2 ratios (CW/OW) of the 12 replicates were calculated for the selected genes and compared with their microarray transcription results. The mean log-2 ratio values > 0 and < 0 indicate genes preferentially transcribed in CW and OW, respectively. Error bars represent the standard deviation of the mean log-2 ratio.