An Early Nodulin-Like Protein Accumulates in the Sieve Element Plasma Membrane of Arabidopsis

Abstract

Membrane proteins within the sieve element-companion cell complex have essential roles in the physiological functioning of the phloem. The monoclonal antibody line RS6, selected from hybridomas raised against sieve elements isolated from California shield leaf (Streptanthus tortuosus; Brassicaceae) tissue cultures, recognizes an antigen in the Arabidopsis (Arabidopsis thaliana) ecotype Columbia that is associated specifically with the plasma membrane of sieve elements, but not companion cells, and accumulates at the earliest stages of sieve element differentiation. The identity of the RS6 antigen was revealed by reverse transcription-PCR of Arabidopsis leaf RNA using degenerate primers to be an early nodulin (ENOD)-like protein that is encoded by the expressed gene At3g20570. Arabidopsis ENOD-like proteins are encoded by a multigene family composed of several types of structurally related phytocyanins that have a similar overall domain structure of an amino-terminal signal peptide, plastocyanin-like copper-binding domain, proline/serine-rich domain, and carboxy-terminal hydrophobic domain. The amino-and carboxy-terminal domains of the 21.5-kD sieve element-specific ENOD are posttranslationally cleaved from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both floral and vegetative tissues, the sieve element-specific ENOD is expressed only within the phloem. Members of the ENOD subfamily of the cupredoxin superfamily do not appear to bind copper and have unknown functions. Phenotypic analysis of homozygous T-DNA insertion mutants for the gene At3g20570 shows minimal alteration in vegetative growth but a significant reduction in the overall reproductive potential.

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

“…At the same time, of course, a wide variety of integral membrane proteins are known and expected to be found in SEs. The target of the SE-specific antibody RS6, raised against the isolated SEs of Streptanthus tortuosus (Brassicaceae), was recently found in Arabidopsis to be an ENOD-like integral protein (Khan et al, 2007). Other proteins have been immunolocalized to SEs, such as the Suc-H 1 symporter SUT1 in potato (Solanum tuberosum; Kü hn et al, 1997), a putative monosaccharide transporter and a protonATPase in developing apple (Malus domestica) fruit (Zhang et al, 2004), a proton-amino acid transporter in Arabidopsis roots (Okumoto et al, 2004), a calcium channel-like protein in tobacco and Pistia stratiotes leaves (Volk and Franceschi, 2000), and a plasma membrane aquaporin in spinach (Spinacia oleracea; Fraysse et al, 2005).…”

“…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.…”

“…Designing SE-specific markers in mature phloem represents a significant challenge. Membrane proteins expressed in immature SEs that are still nucleate, such as the early nodulin (ENOD)-like protein of Arabidopsis (Arabidopsis thaliana; Khan et al, 2007), are likely diluted or degraded as the SEs expand and mature, making them potentially poor targets for fluorescence tagging. Mature SEs lose the capacity for protein expression, delegating it to their sister cells, the companion cells (CCs;van Bel and Knoblauch, 2000).…”

Differential vascularization of nematode-induced feeding sites

“…These data were corroborated by immunocytochemistry. Localization of the SE-specific RS6 antigen (17) characterized the sGFP-labeled cells surrounding the GCs as SEs. These SEs were formed de novo and contained nuclei.…”

Identification and Functional Characterization an Ortholog of OsENOD93-1 Gene in Wheat Using in-silico Approach

“…Seven nodulin-like protein families have been predicted in nonleguminous plant species. Prediction of these families reflects that they probably play an important role in a wide variety of physiological processes (Borner et al, 2003;Khan et al, 2007;Mashiguchi et al, 2009). In rice genome, six ENOD domain containing genes have been reported on same chromosomal region along with similar molecular weight and pI.…”