Structure of the developing pea seed coat and the post-phloem transport pathway of nutrients.

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.

Members of the aquaporin family in the developing pea seed coat include representatives of the PIP, TIP, and NIP subfamilies.

Water and nutrients required by developing seeds are mainly supplied by the phloem and have to be released from a maternal parenchyma tissue before being utilized by the filial tissues of embryo and endosperm. To identify aquaporins that could be involved in this process four full-length cDNAs were cloned and sequenced from a cDNA library of developing seed coats of pea (Pisum sativum L.). The cDNA of PsPIP1-1 appeared to be identical to that of clone 7a/TRG-31, a turgor-responsive gene cloned previously from pea roots. PsPIP1-1, PsPIP2-1, and PsTIP1-1, or their possible close homologues, were also expressed in cotyledons of developing and germinating seeds, and in roots and shoots of seedlings, but transcripts of PsNIP-1 were only detected in the seed coat. In mature dry seeds, high hybridization signals were observed with the probe for PsPIP1-1, but transcripts of PsPIP2-1, PsTIP1-1, and PsNIP-1 were not detected. Functional characterization after heterologous expression in Xenopus oocytes showed that PsPIP2-1 and PsTIP1-1 are aquaporins whereas PsNIP-1 is an aquaglyceroporin. PsNIP-1, like several other NIPs, contains a tryptophan residue corresponding with Trp-48 in GlpF (the glycerol facilitator of Escherichia coli) that borders the selectivity filter in the permeation channel. It is suggested that PsPIP1-1 and/or its possible close homologues could play a role in water absorption during seed imbibition, and that PsPIP2-1, possibly together with PsPIP1-1, could be involved in the release of phloem water from the seed coat symplast, which is intimately connected with the release of nutrients for the embryo.