Jaemo Yang

Plant Body Weight-Induced Secondary Growth in Arabidopsis and Its Transcription Phenotype Revealed by Whole-Transcriptome Profiling

Wood is an important raw material and environmentally cost-effective renewable source of energy. However, the molecular biology of wood formation (i.e. secondary growth) is surprisingly understudied. A novel experimental system was employed to study the molecular regulation of secondary xylem formation in Arabidopsis. First, we demonstrate that the weight carried by the stem is a primary signal for the induction of cambium differentiation and the plant hormone, auxin, is a downstream carrier of the signal for this process. We used Arabidopsis whole-transcriptome (23 K) GeneChip analysis to examine gene expression profile changes in the inflorescent stems treated for wood formation by cultural manipulation or artificial weight application. Many of the genes up-regulated in wood-forming stems had auxin responsive cis-acting elements in their promoter region, indicating auxin-mediated regulation of secondary growth. We identified 700 genes that were differentially expressed during the transition from primary growth to secondary growth. More than 40% of the genes that were up-regulated (>5×) were associated with signal transduction and transcriptional regulation. Biological significance of these regulatory genes is discussed in light of the induction and development of secondary xylem.

Novel gene expression profiles define the metabolic and physiological processes characteristic of wood and its extractive formation in a hardwood tree species, Robinia pseudoacacia.

Wood is of critical importance to humans as a primary feedstock for biofuel, fiber, solid wood products, and various natural compounds including pharmaceuticals. The trunk wood of most tree species has two distinctly different regions: sapwood and heartwood. In addition to the major constituents, wood contains extraneous chemicals that can be removed by extraction with various solvents. The composition and the content of the extractives vary depending on such factors as, species, growth conditions, and time of year when the tree is cut. Despite the great commercial and keen scientific interest, little is known about the tree-specific biology of the formation of heartwood and its extractives. In order to gain insight on the molecular regulations of heartwood and its extractive formation, we carried out global examination of gene expression profiles across the trunk wood of black locust (Robinia pseudoacacia L.) trees. Of the 2,915 expressed sequenced tags (ESTs) that were generated and analyzed in the current study, 55.3% showed no match to known sequences. Cluster analysis of the ESTs identified a total of 2278 unigene sets, which were used to construct cDNA microarrays. Microarray hybridization analyses were then performed to survey the changes in gene expression profiles of trunk wood. The gene expression profiles of wood formation differ according to the region of trunk wood sampled, with highly expressed genes defining the metabolic and physiological processes characteristic of each region. For example, the gene encoding sugar transport had the highest expression in the sapwood, while the structural genes for flavonoid biosynthesis were up-regulated in the sapwood-heartwood transition zone. This analysis also established the expression patterns of 341 previously unknown genes.

Functional Characterization of Allantoinase Genes from Arabidopsis and a Nonureide-Type Legume Black Locust

The availability of nitrogen is a limiting factor for plant growth in most soils. Allantoin and its degradation derivatives are a group of soil heterocyclic nitrogen compounds that play an essential role in the assimilation, metabolism, transport, and storage of nitrogen in plants. Allantoinase is a key enzyme for biogenesis and degradation of these ureide compounds. Here, we describe the isolation of two functional allantoinase genes, AtALN (Arabidopsis allantoinase) and RpALN (Robinia pseudoacacia allantoinase), from Arabidopsis and black locust (Robinia pseudoacacia). The proteins encoded by those genes were predicted to have a signal peptide for the secretory pathway, which is consistent with earlier biochemical work that localized allantoinase activity to microbodies and endoplasmic reticulum (Hanks et al., 1981). Their functions were confirmed by genetic complementation of a yeast mutant (dal1) deficient in allantoin hydrolysis. The absence of nitrogen in the medium increased the expression of the genes. In Arabidopsis, the addition of allantoin to the medium as a sole source of nitrogen resulted in the up-regulation of the AtALN gene. The black locust gene (RpALN) was differentially regulated in cotyledons, axis, and hypocotyls during seed germination and seedling growth, but was not expressed in root tissues. In the trunk wood of a mature black locust tree, the RpALN gene was highly expressed in the bark/cambial region, but had no detectable expression in the sapwood or sapwood-heartwood transition zone. In addition, the gene expression in the bark/cambial region was up-regulated in spring and fall when compared with summer, suggesting its involvement in nitrogen mobilization.

Cloning, characterization, and heterologous expression of a functional geranylgeranyl pyrophosphate synthase from sunflower (Helianthus annuus L.).

Summary Geranylgeranyl pyrophosphate (GGPP) synthase is a key enzyme for the biosynthesis of terpenoid compounds that play vital roles in plant growth and development, and interactions between organisms. We have cloned and characterized a sunflower cDNA encoding GGPP synthase. Sequence analysis showed that the gene contained a 1 071-bp open reading frame coding for a peptide of 356 amino acid residues with a calculated molecular mass of 38.7 kDa. The predicted amino acid sequence of this sunflower GGPS has high similarity to other plant GGPP synthases (63–79% ). In vitro activity assay using recombinant protein and genetic complementation experiments have shown that the cDNA we cloned encodes for functional GGPP synthase. Gene expression studies using sunflower seedlings showed that the gene was expressed after two days of seed imbibition. Abscisic acid treatment down-regulated the expression of the gene.

Molecular cloning and characterization of a functional cDNA clone encoding isopentenyl diphosphate isomerase from Hevea brasiliensis.

Summary Plants produce many isoprenoid compounds of biological and/or economic importance. The first step in isoprenoid biosynthesis is the isomerization of isopentenyl diphosphate (IPP) to its electrophilic isomer, dimethylallyl diphosphate (DMAPP), by the enzyme IPP isomerase (EC 5.3.3.2). The following successive head-to-tail condensation reactions of the five-carbon intermediates lead to the synthesis of a variety of isoprenoids. Among these polyisoprenes, natural rubber is an important raw material with many industrial uses. In order to build our knowledge base of rubber biosynthesis, we cloned and characterized two cDNA clones encoding the IPP isomerase from the latex of Hevea brasiliensis , where a large quantity of natural rubber ( cis -1,4-polyisoprene) is produced. The two clones differ only in the untranslated regions, and have a continuous open reading frame encoding a peptide of 234 amino acids with a predicted molecular mass of 26.7 kDa. The deduced protein is acidic, with an isoelectric point of 4. 7, and shows high sequence identity with other IPP isomerases. The recombinant protein expressed in Escherichia coli showed IPP isomerase activity. In vitro rubber biosynthesis assays using washed rubber particles deprived of initiating allylic diphosphates, revealed that the recombinant IPP isomerase is functional in catalyzing the conversion of IPP to DMAPP, a key activation step of the basic five-carbon isoprene unit in isoprenoid biosynthesis. Southern analysis indicated that the IPP isomerase is encoded by two genes in Hevea rubber tree. Analyses of RNA extracted from extruded latex of the trees, wounded with nails, showed that wounding did not change the transcript level of IPP isomerase.

A shoot regeneration protocol effective on diverse genotypes of sunflower (Helianthus Annuus L.)

SummaryWe describe a protocol, and several experiments that helped lead to its development, for sunflower regeneration. Important factors for sunflower regeneration were explant age, cytokinin type and concentration, basal medium, and explant source. We could not induce shoot regeneration from the explants derived from mature tissues including leaf, petiole, and stem. However, use of juvenile explants such as embryo meristem and primordial leaf tissues allowed routine regeneration of 17 different sunflower genotypes. High frequency of shoot regeneration was achieved with these explants taken from seedlings up to 5 d after germination. Explant age was less critical for embryo meristem explants than for primordial leaf tissues. Of the four basal media tested, MS and B5 media produced higher shoot-regeneration frequencies than did Anderson and woody plant media. The highest shoot-regeneration frequency was obtained with MS medium supplemented with 2 μM BA and without auxin. Addition of 1 μM naphthalene-acetic acid to the medium significantly reduced both the percentage of explants producing shoots and average number of shoots per explant. Regenerated shoots were grown to maturity in a greenhouse.

Functional Genomics of Wood Formation

Wood is of primary importance to humans as timber for construction, and wood-pulp for paper manufacturing. It is also the most environmentally costeffective renewable source of energy. Resolving the dilemma of preserving forest ecosystems, while meeting the increasing demand of forest utilization, necessitates gaining a fundamental understanding of the biochemical processes involved in tree growth and development.