The woody tissues of angiosperm trees, the xylem fibers and vessels, are formed from the lateral meristem of the stem, the vascular cambium. In contrast to vessel elements, which differentiate very rapidly close to the vascular cambium, fiber differentiation is a relatively slow process involving initial expansion of the cells in both the radial and longitudinal dimensions, followed by extensive synthesis of the secondary cell walls. The final phase in maturation of both vessel elements and fibers is cell death and autolysis of the cell contents.
Xylem-cell death involves a range of morphological and nuclear changes in a strictly spatially and temporally coordinated and programmed fashion [1
]. The programmed cell death (PCD) of xylem has been analyzed in detail in an in vitro
system of Zinnia elegans
, in which mesophyll cells of Zinnia
transdifferentiate into xylem vessels commonly called as tracheary elements in a semi-synchronized manner [3
]. In Zinnia
cells, irreversible differentiation into tracheary elements is marked by the accumulation of hydrolytic enzymes in the vacuole and deposition of the secondary cell walls, followed by tonoplast disruption, release of the vacuolar proteases and nucleases into the cytoplasm, and finally the autolytic loss of cell contents [2
]. Several different types of proteases have been detected in Zinnia
], and an S1-type nuclease, capable of hydrolyzing both DNA and RNA, seems to control nuclear DNA degradation in the Zinnia
tracheary elements [7
]. Even though the chain of events during tracheary element PCD is well characterized in the Zinnia
system, very little is known about the regulation of this process in intact plants. In addition, the Zinnia
system has not allowed analysis of the different cell types of the xylem, such as the fibers.
Programmed cell death also occurs in plants in response to external factors, such as avirulent pathogens, giving rise to the so-called hypersensitive response (HR) and in response to shortening daylength - manifested in the senescence of leaves. HR cell death is usually fast and it shares certain features with the apoptotic death of animal cells, such as nuclear shrinkage and fragmentation of DNA into oligonucleosomal multiples of 180-bp fragments [8
]. Senescence-induced cell death is a much slower process, involving nuclear degradation, DNA fragmentation and thorough proteolytic degradation of the cellular contents and controlled remobilization of the nutrients [9
]. The death of the xylem elements is different from HR and senescence-related PCD in that the organellar structure remains intact until vacuolar collapse and the oligonucleosomal DNA fragmentation does not precede cell death [1
]. Whether these processes are related at the molecular level is unknown, but the differences in temporal and spatial regulation, and in cellular morphology, suggest that there are significant differences not only in the early regulation, but also in the execution of the various plant PCD processes.
The genus Populus
has emerged as the main model system for trees, because of its amenability for genomic and molecular analyses [10
is also suitable for analysis of xylem development [11
]. A Populus
expressed sequence tag (EST) database (POPULUSDB) was created from 19 different cDNA libraries [13
]. The database consists of 102,019 ESTs, assembled into a unigene set of 11,885 clusters and 12,759 non-clustered singletons corresponding altogether to 24,644 unique sequences or transcripts [14
]. The great diversity of the tissue types giving rise to the different cDNA libraries enables digital analysis of gene expression by comparison of the EST frequencies in the different libraries. One of the libraries was produced from Populus
woody tissues composed of xylem fibers undergoing cell death. In this work, we studied gene expression in the process of fiber death by in silico
analysis of this 'fiber death library' and by a microarray analysis with a novel Populus
25K cDNA microarray. In addition to its economic importance as one of the processes that regulate wood quality, fiber-cell death is an interesting biological process that as yet is poorly understood. Our analysis identified several novel candidate regulatory genes for xylem PCD.