Taku Demura

Novel vascular cell-specific genes whose expression is regulated temporally and spatially during vascular system development.

We have isolated three cDNA clones (TED2, TED3, and TED4) for genes expressed preferentially in cells that redifferentiate into tracheary elements from mesophyll cells isolated from leaves of Zinnia elegans. Sequence analyses of TED clones revealed that TED2 encodes a hydrophobic polypeptide with a significant similarity to the guinea pig lens-specific protein (zeta-crystallin) and that the deduced polypeptide of TED3 may be a novel cell wall protein. In situ hybridization of the TED probes with young Zinnia seedlings showed that expression of the three TED genes was restricted to vascular cells and regulated in a temporal and spatial manner during vascular development. TED3 transcripts were localized specifically to a few cells that are to differentiate or are differentiating into tracheary elements in all organs examined. TED4 transcripts were present mainly in the immature primary xylem both of cotyledons and of the boundary region between the root and hypocotyl and in the procambium of roots. In contrast, TED2 transcripts accumulated not only in immature primary xylem cells but also in immature phloem cells both in roots and in the boundary region between the root and hypocotyl. In addition, TED2 transcripts were expressed in the procambium cells of roots. In cotyledons, TED2 transcripts did not accumulate in xylem or phloem cells but only in two regions that might form a new vein just outside the phloem of the main leaf vein. Taken together, our findings indicate that TED2, TED3, and TED4 can be novel and efficient markers for development of the vascular system.

Molecular Cloning and Characterization of cDNAs Associated with Tracheary Element Differentiation in Cultured Zinnia Cells

Mesophyll cells isolated mechanically from leaves of Zinnia elegans L. cv Canary bird differentiate into tracheary elements (TE) semisynchronously and at high frequency. Using this system, three cDNA clones, TED2 to TED4, whose corresponding mRNAs were expressed in a close association with tracheary element differentiation, were isolated by differential screening of [lambda]gt11 cDNA library. The library was prepared using poly(A)+ RNA from cells cultured in a TE-induced medium for 48 h prior to morphological changes, including secondary cell-wall thickenings and autolysis. Northern analysis indicated that mRNAs corresponding to the clones were expressed preferentially in cells differentiating into TEs prior to the morphological changes. The expression of the mRNAs was found not to be induced by [alpha]-naphthaleneacetic acid or benzyladenine solely and not to be associated directly with cell division. Analysis of the nucleotide sequence of TED4 showed that the cDNA contains an open reading frame of 285 bp, encoding a polypeptide comprising 95 amino acid residues with a predicted molecular mass of 10.0 kD. A homology search of the nucleotide and amino acid sequences of TED4 with several data bases revealed a significant similarity to those of the barley aleurone-specific clone B11E, which was isolated as an aleurone-specific cDNA from 20-d postanthesis grain.