An important function of the seed coat is to deliver nutrients to the embryo. To relate this function to anatomical characteristics, the developing seed coat of pea (Pisum sativum L.) was examined by light- and cryo-scanning electron microscopy (cryo-SEM) from the late pre-storage phase until the end of seed filling. During this time the apparently undifferentiated seed coat tissues evolve into the epidermal macrosclereids, the hypodermal hourglass cells, chlorenchyma, ground parenchyma and branched parenchyma. Using the fluorescent symplast tracer 8-hydroxypyrene-1,3,6-trisulfonic acid, it could be demonstrated that solutes imported by the phloem move into the chlorenchyma and ground parenchyma, but not into the branched parenchyma. From a comparison with literature data of common bean (Phaseolus vulgaris L.) and broad bean (Vicia faba L.), it is concluded that in the three species different parenchyma layers, but not the branched parenchyma, may be involved in the post-phloem symplasmic transport of nutrients in the seed coat. In pea, the branched parenchyma dies during the storage phase, and its cell wall remnants then form the boundary layer between the living seed coat parenchyma cells and the cotyledons. Using cryo-SEM, clear images were obtained of this boundary layer which showed that many intracellular spaces in the seed coat parenchyma are filled with an aqueous solution. This is suggested to facilitate the diffusion of nutrients from the site of unloading towards the cotyledons.

In the leaves of dicotyledons two modes of phloem loading have been identified (Turgeon and Wimmers, 1988; van Bel et al., 1992van Bel et al., , 1994. These mechanisms of phloem loading, symplasmic or apoplasmic, seem to be associated with minor vein configuration (Gamalei, 1985(Gamalei, , 1989) and carbohydrate metabolism (Gamalei, 1985;Turgeon et al., 1993;Flora and Madore, 1996). CCs in minor veins of apoplasmically phloem-loading species (further referred to as apoplasmic species) are termed TCs and have virtually no plasmodesmata at the interface with the mesophyll domains (Gamalei, 1989). The TCs possess cell wall protrusions, varying in surface area with the transit of photosynthate, and unfragmented vacuoles (Gamalei, 1989; Wimmers and Turgeon, 1991;Gamalei et al., 1992). CCs in minor veins of symplasmically phloem-loading species (further referred to as symplasmic species) are termed ICs and are connected with the mesophyll symplast via numerous plasmodesmata (Gamalei, 1989). The ICs usually contain vesicular networks or heavily fragmented vacuoles and have no cell wall protrusions (Gamalei, 1989).The most persuasive evidence for two phloem-loading mechanisms is the consistent coincidence between physiological behavior and minor vein configuration. The diverse structure-functional indications in favor of two modes of phloem loading are numerous and were thoroughly reviewed by van Bel (1996). More recent evidence supports the existence of principally different systems of phloem loading (Flora and Madore, 1996;Kingston-Smith and Pollock, 1996). The question arises whether the different ways of carbohydrate processing in the phloem-loading zone continue to exist along the phloem trajectory, of which ultrastructure and plasmodesmal connectivity of the CCs in the transport phloem may be indicative. Hence, the present electronmicroscopic investigation was focused on the ultrastructure of the CCs in the transport phloem of two species that were described to be symplasmic, squash (Cucurbita maxima L.;Gamalei, 1991) and Lythrum salicaria L. (van Bel et al., 1994), and two species that were considered to be apoplasmic, broad bean (Vicia faba L.; Gamalei, 1991) and Zinnia elegans L. (Y.V. Gamalei and A.V. Sjutkina, unpublished results), to obtain an impression of the functional continuity between collection and transport phloem in apoplasmic and symplasmic loaders. MATERIALS AND METHODSPhloem specimens were cut from the stems of four species, broad bean (Vicia faba L. cv Witkiem major [Nunhems Zaden bv, Haelen, The Netherlands]), Lythrum salicaria L., Zinnia elegans L. (bv Cruydthoeck, Groningen, The Netherlands), and squash (Cucurbita maxima L. cv Golden Deli-1 A part of this study was subsidized by NWO (Dutch Organization for Scientific Research).

1984. Turgor-sensitive sucrose and amino acid transport into developing seeds of Pisum sativum. Effect of a high sucrose or mannitol concentration in experiments with empty ovules. -Physiol. Plant. 61: 172-182.Sugar and amino acid transport into empty ovules of Pisum sativum L. cv. Marzia was examined. In frtiits containing 4-6 developing seeds, the embryo was removed from four ovules. After this surgical treatment, each empty seed coat was filled with a solution (pH 5.5) containing a low (0, 50 or 200 mM), medium (350,400 or 500^ mM) or high (0.7 or 1 M) concentration of sucrose and/or mannitol. In pulse-labelling experiments with sucrose and a-aminolsobutyric acid (AIB), transport of sucrose and AIB into an empty ovule filled with a solution containing a high sucrose concentration was the same as transport into an ovule filled with a mannitol solution of similar osmolarity, demonstrating that a high sucrose concentration in the seed coat apoplast affects phloem transport of sucrose and AIB into the seed coat only by the osmotic effect. The osmolarity of a given solution filling the seed coat cavity appeared to be important for phloem transport of sucrose and AIB into empty ovules. In our experiments, 350 mM appeared to be the optimal concentration for sucrose and AIB transport into the cavity within an empty ovule, giving results comparable with transport into intact ovules. A lower osmoladty of the solution induced less transport. Very high sucrose or mannitol concentrations caused a strong inhibition of sucrose and AIB unloading from the seed coat, so that transport into the empty ovules was inhibited. A low (strongly negative) but not too low osmotic potential of the solution in tlie seed coat apoplast seems necessary to maintain a normal rate of phloem transport into developing seeds. Apparently, the "sink strength" of developing seeds is turgor-sensitive.Additional key words -Garden pea, phloem transport, phloem unloading, pressureflow mechanism, seed development, sink strength. P. Wolswinket (reprint requests) and A.

Complete submergence represses photosynthesis and aerobic respiration, causing rapid mortality in most terrestrial plants. However, some plants have evolved traits allowing them to survive prolonged flooding, such as species of the genus Rorippa, close relatives of Arabidopsis (Arabidopsis thaliana). We studied plant survival, changes in carbohydrate and metabolite concentrations, and transcriptome responses to submergence of two species, Rorippa sylvestris and Rorippa amphibia. We exploited the close relationship between Rorippa species and the model species Arabidopsis by using Arabidopsis GeneChip microarrays for whole-genome transcript profiling of roots of young plants exposed to a 24-h submergence treatment or air. A probe mask was used based on hybridization of genomic DNA of both species to the arrays, so that weak probe signals due to Rorippa species/Arabidopsis mismatches were removed. Furthermore, we compared Rorippa species microarray results with those obtained for roots of submerged Arabidopsis plants. Both Rorippa species could tolerate deep submergence, with R. sylvestris surviving much longer than R. amphibia. Submergence resulted in the induction of genes involved in glycolysis and fermentation and the repression of many energy-consuming pathways, similar to the low-oxygen and submergence response of Arabidopsis and rice (Oryza sativa). The qualitative responses of both Rorippa species to submergence appeared roughly similar but differed quantitatively. Notably, glycolysis and fermentation genes and a gene encoding sucrose synthase were more strongly induced in the less tolerant R. amphibia than in R. sylvestris. A comparison with Arabidopsis microarray studies on submerged roots revealed some interesting differences and potential tolerance-related genes in Rorippa species.

Plant species were selected on the basis of abundant or no symplasmic continuity between sieveelement-companion-cell (SE-CC) complexes and adjacent cells in the minor veins. Symplasmic continuity and discontinuity are denoted, respectively, as symplasmic and apoplasmic minor-vein configurations. Discs of predarkened leaves from which the lower epidermis had been removed, were exposed to (14)CO2. After 2 h of subsequent incubation, phloem loading in control discs and discs treated with p-chloromercuribenzenesulfonic acid (PCMBS) was recorded by autoradiography. Phloem loading was strongly suppressed by PCMBS in minor veins with symplasmically isolated SE-CC complexes (Centaurea, Impatiens, Ligularia, Pelargonium, Pisum, Symphytum). No significant inhibition of phloem loading by PCMBS was observed in minor veins containing sieve elements with abundant symplasmic connections (Epilobium, Fuchsia, Hydrangea, Oenothera, Origanum, Stachys). Phloem loading in minor veins with both types of SE-CC complex (Acanthus) had apoplasmic features. The results provide strong evidence for coincidence between the mode of phloem loading and the minor-vein configuration. The widespread occurrence of a symplasmic mode of phloem loading is postulated.