Richard D. Sjolund

The Phloem Sieve Element: A River Runs through It.

31 9-335-3620. the sieve tube, which is formed by a series of connected phloem sieve elements. This fundamental difference imposes requirements on the development of the sieve element that are unique to these cells, and the differentiation of sieve elements follows a course that is unparalleled in the biological world. Essentially, a series of differentiating sieve elements form a syncytium, a single compartment bound by a plasma membrane (Murphy and Aikman, 1989). Within the developing syncytium, the larger cellular organelles, which could impede assimilate flow, are degraded and removed, and the mature sieve tube lacks nuclei, vacuoles, Golgi bodies, or ribosomes. The plasmodesmata in the walls between the individual sieve elements are converted into large openings (sieve pores) that facilitate flow from cell to cell through the sieve tube. Significantly, this differentiation process, including the formation of sieve pores, is accomplished while maintaining the continuity of the plasma membrane around the individual cells and through the pores formed between the cells. The result of this unique differentiation process is the formation of a continuous, membrane-lined compartment, the sieve tube, that provides a pathway for nutrient flow and for signaling activities (Ryals et al., 1996) throughout the plant body.

Identification and characterization of a phloem-specific beta-amylase.

A monoclonal antibody, RS 5, was raised by injecting sieve elements isolated from tissue cultures of Streptanthus tortuosus (Brassicaceae) into BALB/c mice and screening resultant hybridoma supernatants for the labeling of phloem using immunofluorescence microscopy. The RS 5 monoclonal antibody identifies a 57-kD protein on immunoblots, which is present in phloem-forming tissue cultures of S. tortuosus but is absent in cultures that lack phloem. Purified 57-kD protein of S. tortuosus is demonstrated to be a phloem-specific [beta]-amylase. Partial peptide sequences of the 57-kD protein of S. tortuosus are shown to be 96% identical with the corresponding portions of a deduced sequence reported for a major form of [beta]-amylase in Arabidopsis thaliana. The RS 5 antibody cross-reacts with the major form of A. thaliana [beta]-amylase on immunoblots, and the antibody also binds to the sieve elements of A. thaliana using immunofluorescence microscopy. The results suggest that the major form of A. thaliana [beta]-amylase is a phloem-specific enzyme.

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

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.

Transport of glucose, fructose and sucrose by Streptanthus tortuosus suspension cells. II: Uptake at high sugar concentration

Streptanthus tortuosus Kell. suspension cells will grow in a medium with sucrose as carbohydrate source. It was investigated whether the cells are able to take up sucrose or whether sucrose has to be hydrolyzed to glucose and fructose which eventually are taken up. The detailed quantitative analysis of sugar-uptake rates in the low concentration range up to 1 mM showed the following features: (i) There is definitely no sucrose-uptake system working in the low concentration range; any uptake of radioactivity from labelled sucrose proceeds via hydrolysis of sucrose by cell-wallbound invertase. (ii) Hexoses are taken up by two systems, a glucose-specific system with a Km of 45 μM and a high Vmax for glucose and a Km of 6 mM and a low Vmax for fructose, and a fructosespecific system with a Km of 500 μM and high a Vmax for fructose and a Km of 650 μM and a low Vmax for glucose. (iii) There is a more than tenfold preference for uptake of the fructose derived from sucrose versus uptake of free fructose, with the result that the kinetic disadvantage of the fructoseuptake system compared to the glucose-uptake system is diminished if sucrose is supplied as the carbon source. It is speculated that invertase might work as an enzyme aiding in fructose transport.

Phloem in Plant Tissue Cultures

In his 1950 paper (Ball 1950), Ball commented that; “The advantages of the use of the sterile culture method for study of differentiation in the tissues of vascular plants have not been fully utilized.” Forty five years later, his observation can probably still be applied to the study of phloem tissues using in vitro techniques, but certainly not to the study of xylem using similar approaches. This difference in progress between the study of phloem and xylem using cultured cells is the result of several factors.

Sieve Elements Isolated from Callus Cultures Can be used to Raise Phloem-Specific Monoclonal Antibodies

Phloem sieve elements function in phloem loading in vitro and can be separated from callus cultures of Streptanthus tortuosus and Arabidopsis thaliana by cell wall digestion, filtration and density gradient centrifugation. Isolated sieve elements have been used to immunize mice and to raise monoclonal antibodies that recognize phloem cells in the callus cultures and in organs of the parent plants. The monoclonal antibodies also identify unknown proteins on immunoblots, providing a technique for the identification of novel antigens from cell cultures based on cell specificity and immuno-microscopy.