Details

Autor(en) / Beteiligte

• Avci, Utku

Titel

Electron microscopy complements genetic manipulation for understanding xylem development

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2007

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Availability of genome sequences, and gene-specific mutant collections has made genetic approaches, including gene knockout and over-expression, faster and easier ways to characterize functions of genes related to specific biological processes. Among the methods used towards identification of gene functions, electron microscopy, which requires a dedicated person and special skills, is generally neglected. This research mainly utilized electron microscopy techniques and proved how this approach can reveal key results, otherwise impossible to observe. Sample preparation of Arabidopsis for electron microscopic observation was aided by fast microwave-assisted processing of whole seedlings. This method: (a) yielded good ultrastructural preservation even with mild fixation to retain protein antigenicity in immunolabeling protocols; and (b) minimized potential artifacts of specimen handling and movement of soluble proteins. The research focused on tracheary elements (TEs), which are the principal conductive cells of the xylem. Tracheary elements, including vessels and tracheids, function to deliver water throughout plant parts and give mechanical strength to the plant body. Tracheary elements are differentiated from procambium or vascular cambium through cell changes including secondary wall synthesis, cell suicide and autolysis. This research revealed key results in many aspects of above-mentioned differentiation process. Ultrastructural observations reported here showed the details of tonoplast implosion and a new final stage of TE autolysis. Ultrastructural details of cells and cell walls were determined upon over-expression of a transcription factor and a cellulose synthase gene. Genetic manipulations related to three aspects of vascular cell differentiation were analyzed. First, the cellular roles of two xylem-specific cysteine proteases from Arabidopsis thaliana, XCP1 and XCP2, were analyzed in root primary xylem. These proteases have been hypothesized to be involved in TE autolysis, a process essential to the creation of hollow water-conducting xylem vessels. This research provided direct support for this hypothesis and new insights into TE autolysis and the cellular behavior of cysteine proteases. XCP1 and XCP2 were only detected in the cytoplasm until a late stage of differentiation when they were visualized within the central vacuole along with cellular components destined to be degraded there. Analysis of T-DNA lines showed that xcp1 and xcp1xcp2 TEs had cellular remnants that persisted after wild type TEs had fully cleared. Therefore, XCP1 degrades particular cellular components during TE autolysis, and, as suggested by the different appearance of cellular remnants in xcp1 vs. xcp1xcp2 TEs, XCP2 is likely to have a related but distinct role. Second, the cellular role of Xylem NAC Domain1 (XND1), a transcription factor, was investigated during Arabidopsis primary xylem differentiation. XND1 is highly expressed and regulated in xylem. Over-expression of XND1 in Arabidopsis resulted in extreme dwarfism and blocked xylem differentiation. Electron microscopy showed that the incipient xylem cells failed to deposit patterned secondary walls, and they did not undergo autolysis. The phloem cells in XND1 over-expressors proliferated and had highly thickened cell walls that appeared as an exaggeration of the phloem wall structure seen in the control plants. These dwarf plants also had unusual poorly developed plastids that were frequently seen in the cortex cells. Third, cellulose synthases (CesAs) are major, highly regulated players in cellulose biosynthesis although their exact biochemical role is still unknown. Over-expression in aspen of a previously cloned CesA gene from aspen (PtrCesA1) resulted in reduction in the amount of stem cellulose as determined chemically elsewhere. Through light and electron microscopic observations, this research showed collapse in the overall tissue of secondary xylem in over-expressors as well as reduced secondary wall birefringence in the polarizing microscope. Changes in the middle lamella and layering in the secondary wall were also detected.