Co-expression Analysis of Genes Associated with Cambial Cell Differentation during Wood Formation

Abstract

Wood is common name of xylem of trees, an important source of fixed carbon used for woody materials and industrial purposes such as timber, pulp, furniture, fibers, and also as energy source or for other products (films, adhesives, etc). During wood formation cambial daughter cells develop and specialize to xylem cells. The mechanism of wood formation based on the observation of cell structures and cell wall components is well understood, but the genetic control of cambial activity and differentiation is still little known. A recent study in the model tree poplar showed evidences for an involvement of KNOX genes in controlling differentiation of cambial daughter cells. High resolution transcript analyses of the poplar cambium showed several KNOX genes with strong cambial expression (Hertzberg et al. 2001; Schrader et al. 2004). Furthermore, the current understanding of the regulation of differentiation in vascular development was greatly enhanced by the study of the poplar KNOX gene ARBORKNOX1 (ARK1) and ARBORKNOX2 (ARK2), which are close homologues of the Arabidopsis STM and BREVIPEDICELLUS(BP/KNAT1), respectively. ARK1 was shown to be expressed in the cambium and over-expression of ARK1 leads to inhibition of differentiation of vascular cells (Groover et al. 2006). A co-expression analysis of publicly available microarray data was performed in order to identify genes which act downstream of Arabidopsis KNAT1 and STM, using Arabidopsis Co-expression Tool (ACT; www.arabidopsis.leedsac.uk/ACT). Genes, which are positively regulated by KNAT1 or STM should be co-expressed with both of them. From 100 genes co-expressed with either STM or KNAT1, 69 genes (69%) were identical. In other words, those 69 genes are co-expressed with STM and also KNAT1. This astonishingly high overlap underlines the redundant function of STM and KNAT1. Of 69 overlapping genes seven genes were selected based on their association with cambial cell and secondary cell wall formation and their ranking of co-expression. Quantitative expression analysis in wild-type, stm-GK, knat1bp-9 and the double mutant was subsequently performed for the selected genes. Down-regulation of STM and KNAT1 was always followed by a not significant trend of downregulation of cellulose synthases (IRX1, IRX3 and IRX5), cobra-like 4 (IXR6), pectin methylesterase61 (PME61), and fasciclin-like arabinogalactan 11(FLA11) in the single mutants. In the double mutant the down-regulation for all thosegenes was greater than 10 times and highly significant (Table 5). Only the lipid transfer protein 4 (LTP4) behaved in an opposite manner and was upregulated in the double mutant. Thus, STM and KNAT1 are upstream of IRX1, IRX3, IXR6, PME61 and FLA11. To address the potential involvement of STM and KNAT1 in lignin deposition during secondary cell wall formation, key-genes of lignin biosynthesis previously identified by Mele et al (2003) were tested for their expression in the mutants. Interestingly, the expression of At4CL1, PAL1, CAD1, and PRX in stm-GK, knat1bp-9, and stm-GK;knat1bp- 9 was not different from wild type (Col-0). In contrast to cellulose biosynthesis, this may indicate that STM and KNAT1 are not directly involved in lignin biosynthesis.