Kyung Hwan Han

Ectopic expression of MYB46 identifies transcriptional regulatory genes involved in secondary wall biosynthesis in Arabidopsis

MYB46 functions as a transcriptional switch that turns on the genes necessary for secondary wall biosynthesis. Elucidating the transcriptional regulatory network immediately downstream of MYB46 is crucial to our understanding of the molecular and biochemical processes involved in the biosynthesis and deposition of secondary walls in plants. To gain insights into MYB46-mediated transcriptional regulation, we first established an inducible secondary wall thickening system in Arabidopsis by expressing MYB46 under the control of dexamethasone-inducible promoter. Then, we used an ATH1 GeneChip microarray and Illumina digital gene expression system to obtain a series of transcriptome profiles with regard to the induction of secondary wall development. These analyses allowed us to identify a group of transcription factors whose expression coincided with or preceded the induction of secondary wall biosynthetic genes. A transient transcriptional activation assay was used to confirm the hierarchical relationships among the transcription factors in the network. The in vivo assay showed that MYB46 transcriptionally activates downstream target transcription factors, three of which (AtC3H14, MYB52 and MYB63) were shown to be able to activate secondary wall biosynthesis genes. AtC3H14 activated the transcription of all of the secondary wall biosynthesis genes tested, suggesting that AtC3H14 may be another master regulator of secondary wall biosynthesis. The transcription factors identified here may include direct activators of secondary wall biosynthesis genes. The present study discovered novel hierarchical relationships among the transcription factors involved in the transcriptional regulation of secondary wall biosynthesis, and generated several testable hypotheses.

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.

Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers of Hevea brasiliensis (para rubber tree)

Latex of Hevea brasiliensis (Willd. ex A, Juss.) Mull. Arg. (Brazilian rubber tree) contains 30–50% (w/w) of natural rubber (cis-1,4-polyisoprene), which is an important raw material for many industrial uses. In order to gain insights into the molecular events occurring in latex, we analyzed more than 20,000 cDNA-AFLP-based TDFs (transcription-derived fragments) and 1176 ESTs. The results revealed several novel features of the latex transcriptome. First, the repertoire of the genes expressed in latex is unique. Only seven gene families accounted for more than 51% of the latex transcriptome. Among them, two of the most abundant ESTs were the genes encoding rubber particle proteins REF (rubber elongation factor) and SRPP (small rubber particle protein), comprising 29% of the total ESTs. Unexpectedly, several genes involved in the rubber biosynthesis were expressed at low levels in the latex. In fact, genes encoding cis-prenyltransferase (CPT), a potential candidate for rubber polymerase, were not present in the EST pool because of their low expression level. However, we were able to clone four full-length cDNAs by screening the same latex cDNA library used in the EST analysis and confirmed their enzyme activity in vitro. The second most abundant transcripts were defense- or stress-related genes, suggesting that defense is one of the functions of laticifers. Finally, the presence of the non-mevalonate DXP/MEP pathway for IPP synthesis in latex was noted by up-regulation of the 1-deoxy-D-xylulose 5-phosphate synthase gene.

Arabidopsis whole-transcriptome profiling defines the features of coordinated regulations that occur during secondary growth.

Secondary growth in the inflorescence stems of Arabidopsisplants was induced by a combination of short-day and long-day treatments. The induced stems were divided into three different stem developmental stages (i.e., immature, intermediate, and mature) with regard to secondary growth. Whole transcriptome microarrays were used to examine the changes in global gene expression occurring at the different stem developmental stages. Over 70% of the Arabidopsis transcriptome was expressed in the stem tissues. In the mature stems with secondary growth, 567 genes were upregulated 5-fold or higher and 530 were downregulated, when compared to immature stems (with no secondary growth) and 10-day old seedlings (with no inflorescence stem). The transcription phenotypes obtained from the stems at different developmental stages largely confirm the existing insights into the biochemical processes involved in the sequential events that lead to wood formation. The major difference found between the stems undergoing secondary growth and only primary growth was in the expression profiles of transcriptional regulation-and signal transduction-related genes. An analysis of several shoot apical meristem (SAM) activity-related gene expression patterns in the stems indicated that the genetic control of secondary meristem activity might be governed by a different mechanism from that of SAM. The current study established the expression patterns of many unknown genes and identified candidate genes that are involved in the genetic regulation of secondary growth. The findings described in this report should improve our understanding of the molecular mechanisms that regulate the growth and development of the stem.

An update on the nomenclature for the cellulose synthase genes in Populus

Cellulose synthase (CesA) is a central catalyst in the generation of the plant cell wall biomass and is, therefore, the focus of intense research. Characterization of individual CesA genes from Populus species has led to the publication of several different naming conventions for CesA gene family members in this model tree. To help reduce the resulting confusion, we propose here a new phylogeny-based CesA nomenclature that aligns the Populus CesA gene family with the established Arabidopsis thaliana CesA family structure.

Global comparative transcriptome analysis identifies gene network regulating secondary xylem development in Arabidopsis thaliana

Our knowledge of the genetic control of wood formation (i.e., secondary growth) is limited. Here, we present a novel approach to unraveling the gene network regulating secondary xylem development in Arabidopsis, which incorporates complementary platforms of comparative-transcriptome analyses such as “digital northern” and “digital in situ” analysis. This approach effectively eliminated any genes that are expressed in either non-stem tissues/organs (“digital northern”) or phloem and non-vascular regions (“digital in situ”), thereby identifying 52 genes that are upregulated only in the xylem cells of secondary growth tissues as “core xylem gene set”. The proteins encoded by this gene set participate in signal transduction, transcriptional regulation, cell wall metabolism, and unknown functions. Five of the seven signal transduction-related genes represented in the core xylem gene set encode the essential components of ROP (Rho-related GTPase from plants) signaling cascade. Furthermore, the analysis of promoter sequences of the core xylem gene set identified a novel cis-regulatory element, ACAAAGAA. The functional significances of this gene set were verified by several independent experimental and bioinformatics methods.

MYB46 directly regulates the gene expression of secondary wall-associated cellulose synthases in Arabidopsis

Cellulose is the most abundant biopolymer on Earth. Three cellulose synthases (CESA4, CESA7 and CESA8) are necessary for cellulose production in the secondary cell walls of Arabidopsis. Little is known about how expression of these CESA genes is regulated. We recently identified a cis-regulatory element (M46RE) that is recognized by MYB46, which is a master switch for secondary wall formation in Arabidopsis. A genome-wide survey of promoter sequences for the presence of M46REs led to the hypothesis that MYB46 may function as a direct regulator of all three secondary wall-associated cellulose synthase genes: CESA4, CESA7 and CESA8. We tested this hypothesis using several lines of experimental evidence. All three CESA genes are highly up-regulated by both constitutive and inducible over-expression of MYB46 in planta. Using a steroid receptor-based inducible activation system, we show that MYB46 directly activates transcription of the three CESA genes. We then used an electrophoretic mobility shift assay and chromatin immunoprecipitation analysis to confirm that MYB46 protein directly binds to the promoters of the three CESA genes both in vitro and in vivo. Furthermore, ectopic up-regulation of MYB46 resulted in a significant increase of crystalline cellulose content in Arabidopsis. Taken together, we have identified MYB46 as a transcription factor that directly regulates all three secondary wall-associated CESA genes. Yeast one-hybrid screening identified additional transcription factors that regulate the CESA genes. However, none of the putative regulators appears to be regulated by MYB46, suggesting the multi-faceted nature of transcriptional regulation of secondary wall cellulose biosynthesis.

Identification of a cis-acting regulatory motif recognized by MYB46, a master transcriptional regulator of secondary wall biosynthesis

While many aspects of primary cell wall have been extensively elucidated, our current understanding of secondary wall biosynthesis is limited. Recently, transcription factor MYB46 has been identified as a master regulator of secondary wall biosynthesis in Arabidopsis thaliana. To gain better understanding of this MYB46-mediated transcriptional regulation, we analyzed the promoter region of a direct target gene, AtC3H14, of MYB46 and identified a cis-acting regulatory motif that is recognized by MYB46. This MYB46-responsive cis-regulatory element (M46RE) was further characterized and shown to have an eight-nucleotide core motif, RKTWGGTR. We used electrophoretic mobility shift assay, transient transcriptional activation assay and chromatin immunoprecipitation analysis to show that the M46RE was necessary and sufficient for MYB46-responsive transcription. Genome-wide analysis identified that the frequency of M46RE in the promoters were highly enriched among the genes upregulated by MYB46, especially in the group of genes involved in secondary wall biosynthesis.

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.