Protophloem Differentiation in Early Arabidopsis thaliana Development

Abstract

During Arabidopsis embryogenesis, procambial cells undergo coordinated, asymmetric cell divisions, giving rise to vascular precursor cells (protophloem and protoxylem precursors). After germination, these cells terminally differentiate into specialized conducting cells, referred to as protophloem and protoxylem cells. Few readily identifiable markers of the onset of specification and differentiation are available, hampering the molecular genetic analysis of protophloem development. Confocal microscopy was used to investigate the patterning and differentiation of phloem cells during early plant development. Longitudinal divisions of phloem initials allowed the identification of protophloem precursor cells and adjacent metaphloem initials along the length of the plant. During germination, protophloem differentiation was observed at two independent locations, in the cotyledons and the hypocotyl. In both locations, differentiation was concomitant with cell elongation. We identified five gene-trap lines (PD1-PD5) with marker gene expression in immature protophloem elements. The spatio-temporal marker expression pattern of the lines divides them into two groups. The early specification markers PD4 and PD5 were expressed in developing organs before procambium formation and then became restricted to phloem initial cells. The protophloem precursor markers PD1-PD3 were expressed in differentiating protophloem cells at different stages of their development. All markers were expressed transiently and iteratively during the differentiation of protophloem in newly formed organs. Flanking genes were identified for four out of five gene-trap insertion lines. The possible function of these genes with respect to phloem differentiation is discussed.

Arabidopsis NPCC6/NaKR1 Is a Phloem Mobile Metal Binding Protein Necessary for Phloem Function and Root Meristem Maintenance

“…No expression was detected in developing embryos or in seeds before germination. Therefore, NaKR1 is not expressed in the earliest protophloem cells that differentiate in mature embryos (Bauby et al, 2007). By 2 DAG, NaKR1 expression was observed in vascular tissue throughout the seedlings.…”

Dynamic, auxin-responsive plasma membrane-to-nucleus movement ofArabidopsisBRX

“…Indeed, analysis of pBRX::GUS expression in embryos revealed that BRX is ubiquitously expressed at early stages and becomes restricted to the (incipient) vasculature in the mature embryo and at later stages of (adult) development (Fig. 1H-K) (Bauby et al, 2007).…”

A Plasma Membrane-Anchored Fluorescent Protein Fusion Illuminates Sieve Element Plasma Membranes in Arabidopsis and Tobacco

“…Higher resolution imaging of aniline blue-stained plates using a UV laser on a single-photon confocal microscope leads to rapid aniline blue bleaching, but imaging could be possible with lower energy multiphoton microscopy (A. Schulz, personal communication), deconvolution methods, and the pore number calculation introduced in Figure 6. The Bauby et al, 2007), as well as the cell division zone of the expanding root tip (Beemster and Baskin, 1998), demonstrate an mCitrine signal. Bar 5 100 mm.…”

“…Transmission electron microscopy is a potential alternative (Kü hn et al, 1997;Ehlers et al, 2000;Turgeon et al, 2001) but lacks the three-dimensional perspective needed for good quantitative measurements, it requires more time to implement than light microscopy, and it is inappropriate for statistical sampling of large amounts of tissue. Likewise, exogenously applied fluorochromes (Oparka et al, 1995;Knoblauch and van Bel, 1998;Knoblauch et al, 2001;Martens et al, 2006;Bauby et al, 2007) and sieve element (SE)-specific antibodies, like RS6 (Khan et al, 2007), have proven their utility, but none of the fluorochromes is specific to the phloem and the use of antibodies is expensive and time consuming, lessening the opportunity for fast and direct observation of phloem cells alone.…”

“…Despite the time and cost of the development of stably transgenic plants, the study of phloem anatomy and architecture is likely best and ultimately most easily served by plants that express markers in phloem cells under the control of developmentally or physiologically significant promoters (Imlau et al, 1999;Stadler et al, 2005;Bauby et al, 2007). For present purposes, an ideal marker is one that fluorescently labels the plasma membranes of conductive SEs and sieve pores with an endogenous marker and allows quick measurements of the quantitative geometry of SEs (their radius and length) and sieve plates (the radius and length and number of sieve pores in each sieve plate) down to the resolution limits of light microscopy.…”