Although a substantial proportion of plant biomass originates from the activity of vascular cambium, the molecular basis of radial plant growth is still largely unknown. To address whether cytokinins ...are required for cambial activity, we studied cytokinin signaling across the cambial zones of 2 tree species, poplar (Populus trichocarpa) and birch (Betula pendula). We observed an expression peak for genes encoding cytokinin receptors in the dividing cambial cells. We reduced cytokinin levels endogenously by engineering transgenic poplar trees (P. tremula x tremuloides) to express a cytokinin catabolic gene, Arabidopsis CYTOKININ OXIDASE 2, under the promoter of a birch CYTOKININ RECEPTOR 1 gene. Transgenic trees showed reduced concentration of a biologically active cytokinin, correlating with impaired cytokinin responsiveness. In these trees, both apical and radial growth was compromised. However, radial growth was more affected, as illustrated by a thinner stem diameter than in WT at same height. To dissect radial from apical growth inhibition, we performed a reciprocal grafting experiment. WT scion outgrew the diameter of transgenic stock, implicating cytokinin activity as a direct determinant of radial growth. The reduced radial growth correlated with a reduced number of cambial cell layers. Moreover, expression of a cytokinin primary response gene was dramatically reduced in the thin-stemmed transgenic trees. Thus, a reduced level of cytokinin signaling is the primary basis for the impaired cambial growth observed. Together, our results show that cytokinins are major hormonal regulators required for cambial development.
The developmental ontogeny of the vascular system (consisting of xylem, phloem and procambium) is poorly understood despite its central role in plant physiology. We show that in the Arabidopsis root ...meristem, xylem cell lineages are specified early, whereas phloem and procambium are established through a set of asymmetric cell divisions. These divisions require the WOODEN LEG (WOL) gene. The WOL gene encodes a novel two-component signal transducer with an unusual tandem arrangement of two receiver domains. It is expressed specifically in the vasculature from the early stages of embryogenesis on, consistent with a role as a sensor for vascular morphogenesis.
Wood, or secondary xylem, is a water-conductive and supportive vascular tissue highly characteristic of trees. In addition to parenchymatous cells adapted for storage and transport functions, wood is ...mainly composed of various vertically elongated cell types. These are classified either as tracheary elements or fibers, both of which are characterized with extensive secondary cell wall thickenings. The cell wall characteristics contribute to the properties of wood as a significant raw material for various human applications. Wood formation occurs during the secondary phase of plant development. This results from the activity of the vascular cambium, a lateral meristem that is established and functional during the secondary phase. On the other hand, already the primary phase of vascular development, associated with the procambial development of apical meristems, involves xylem production. The formation of both primary and secondary xylem involves a cascade of interesting processes including specification of primary vascular tissue as bundles, cell proliferation within the primary bundles or in the secondary vascular cambium, initiation of xylem differentiation, regulation of cell expansion, deposition of a secondary cell wall, and programmed cell death. Even as these processes have been extensively documented at the structural level, relatively little is known of the genetic mechanisms behind them. Although wood formation is an evident characteristic of trees, also many herbaceous plants, including Arabidopsis, develop vascular cambium and form secondary xylem. Thus, Arabidopsis can be considered as a model for the developmental processes underlying xylem development during both primary and secondary phases of development. In this Update we will first review the most recent work related to each developmental process resulting in xylem formation and finally focus on the secondary phase of development to compare wood development in Arabidopsis and trees in light of the most recent molecular data.
We have isolated and sequenced cDNA clones that encode human spermine synthase (EC 2.5.1.22). The total length of the sequenced cDNA was 1,612 nucleotides, containing an open reading frame encoding a ...polypeptide chain of 368 amino acids. All of the previously sequenced peptide fragments of human and bovine spermine synthase proteins could be located within the coding region derived from the cDNA. An unusual sequence of AATTAA apparently signaled the initiation of polyadenylation. Sequence comparisons between human spermine synthase and spermidine synthases from bacterial and mammalian sources revealed a nearly complete lack of similarity between the primary structures of these two enzymes catalyzing almost identical reactions. A modest similarity found was restricted to a relatively short peptide domain apparently involved in the binding of decarboxylated S-adenosylmethionine, the common substrate for both enzymes. The apparent lack of an overall similarity may indicate that spermine synthase, the enzyme found only in eukaryotes, and spermidine synthase with more universal distribution, although functionally closely related, have evolved separately.
An increased mRNA content of spermidine synthase was found in phytohemagglutinin stimulated human peripheral lymphocytes and in cultured human myeloma (Sultan) cells stimulated to grow by change of ...the culture medium. The many-fold increase in the amount of the message was accompanied by stimulation of the enzyme activity in activated lymphocytes, but not in stimulated myeloma cells. In the present study the effect of the 5′-untranslated region of spermidine synthase mRNA on the post-transcriptional control of its expression was studied both in vitro in rabbit reticulocyte system and in cultured mammalian cells. The results show that the GC-rich 5′-untranslated region of spermidine synthase mRNA has an inhibitory effect on its translation.
Using a synthetic deoxyoligonucleotide mixture constructed for a tryptic peptide of the bovine enzyme as a probe, cDNA coding for the full-length subunit of spermidine synthase was isolated from a ...human decidual cDNA library constructed on phage lambda gt11. After subcloning into the Eco RI site of pBR322 and propagation, both strands of the insert were sequenced using a shotgun strategy. Starting from the first start codon, which was immediately preceded by a GC-rich region including four overlapping CCGCC consensus sequences, an open reading frame for a 302-amino-acid polypeptide was resolved. This peptide had an Mr of 33,827, started with methionine, and ended with serine. The identity of the isolated cDNA was confirmed by comparison of the deduced amino acid sequence with resolved sequences of the tryptic peptides of bovine spermidine synthase. The coding strand of the cDNA revealed no special regulatory or ribosome-binding signals within 82 nucleotides preceding the start codon and no polyadenylation signal within 247 nucleotides following the stop codon. The coding region, containing a 13-nucleotide repeat close to the 5' end, was longer than, and very different from, that of the bacterial counterpart. This region seems to be of retroviral origin and shows marked homology with sequences found in a variety of human, mammalian, avian, and viral genes and mRNAs. By computer analysis, the first 200 nucleotides of the 5' end of the coding strand appear able to form a very stable secondary structure with a free energy change of -157.6 kcal/mole.
The human spermidine synthase (EC 2.5.1.16) gene was isolated from a genomic library constructed with DNA obtained from a human immunoglobulin G (IgG) myeloma cell line. Subsequent sequence analyses ...revealed that the gene comprised of 5,818 nucleotides from the cap site to the last A of the putative polyadenylation signal with 8 exons and 7 intervening sequences. The 5'-flanking region of the gene was extremely GC rich, lacking any TATA box but containing CCAAT consensus sequences. No perfect consensus sequence for the cAMP-responsive element for the AP-1 binding site was found, yet the gene contained seven AP-2 binding site consensus sequences. The putative polyadenylation signal was an unusual AATACA instead of AATAAA. Polymerase chain reaction analysis with DNA obtained from human x hamster somatic cell hybrids indicated that human spermidine synthase genomic sequences segregate with human chromosome 1. Transfection of the genomic clone into Chinese hamster ovary cells displaying a low endogenous spermidine synthase activity revealed that the gene was transiently expressed and hence in all likelihood represents a functional gene.
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