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

Abstract

Rapid acquisition of quantitative anatomical data from the sieve tubes of angiosperm phloem has been confounded by their small size, their distance from organ surfaces, and the time-consuming nature of traditional methods, such as transmission electron microscopy. To improve access to these cells, for which good anatomical data are critical, a monomeric yellow fluorescent protein (mCitrine) was N-terminally fused to a small (approximately 6 kD) membrane protein (AtRCI2A) and stably expressed in Arabidopsis thaliana (Columbia-0 ecotype) and Nicotiana tabacum (‘Samsun’) under the control of a companion cell-specific promoter (AtSUC2p). The construct, called by its abbreviation SUmCR, yielded stable sieve element (SE) plasma membrane fluorescence labeling, even after plastic (methacrylate) embedding. In conjunction with wide-field fluorescence measurements of sieve pore number and position using aniline blue-stained callose, mCitrine-labeled material was used to calculate rough estimates of sieve tube-specific conductivity for both species. The SUmCR construct also revealed a hitherto unknown expression domain of the AtSUC2 Suc-H+ symporter in the epidermis of the cell division zone of developing root tips. The success of this construct in targeting plasma membrane-anchored fluorescent proteins to SEs could be attributable to the small size of AtRCI2A or to the presence of other signals innate to AtRCI2A that permit the protein to be trafficked to SEs. The construct provides a hitherto unique entrée into companion cell-to-SE protein targeting, as well as a new tool for studying whole-plant phloem anatomy and architecture.

Callose Synthase GSL7 Is Necessary for Normal Phloem Transport and Inflorescence Growth in Arabidopsis

“…Measurements of 20 sieve plates (from at least two stems in each of two separate preparations) gave pore diameters between 0.30 and 0.55 mm. These data accord well with previous estimates for Arabidopsis stems of approximately 0.5 mm (1-week-old stems; Truernit et al, 2008) and 0.52 mm (stems of unspecified age; Thompson and Wolniak, 2008). By contrast, although pore numbers per sieve plate were approximately the same as in wild-type plants, sieve plate pores in stems of mutant plants appeared much smaller in diameter than those in wild-type plants.…”

Transport and Sorting of the Solanum tuberosum Sucrose Transporter SUT1 Is Affected by Posttranslational Modification

“…It is discussed whether the transport of the membrane-anchored protein occurs through the desmotubules connecting the ER network of the companion cells and the sieve elements followed by additional vesicular transport to the sieve element PM. Fluorescence of the membrane-anchored fusion protein is also detected in vesicle-like structures in the mature sieve elements (Thompson and Wolniak, 2008). A similar route is now suggested for the targeting of St SUT1.…”

“…In a recent study, it was shown that expression of a membrane-anchored fluorescent protein under control of the companion cell-specific At SUC2 promoter illuminates the sieve element PM in Arabidopsis and tobacco (Thompson and Wolniak, 2008). It is discussed whether the transport of the membrane-anchored protein occurs through the desmotubules connecting the ER network of the companion cells and the sieve elements followed by additional vesicular transport to the sieve element PM.…”

Phloem-Specific Methionine Recycling Fuels Polyamine Biosynthesis in a Sulfur-Dependent Manner and Promotes Flower and Seed Development

“…Resistant plants were validated visually and with PCR and named MM or DD, respectively. To create the pSUC2::MTI1 and pSUC2:: DEP1 constructs, MTI1 (1125 bp) and DEP1 (1524 bp) coding sequences (CDS) were amplified and inserted into pENTR/D-TOPO, which was recombined with a destination vector containing a 2125-bp-long fragment of the SUC2 promoter (pSUC2-dest; Thompson and Wolniak, 2008). The pSUC2::MTI1 and pSUC2::DEP1 constructs were introduced into mti1-1 and dep1-1 plants by floral dip, respectively, yielding the lines SM and SD.…”