Systematic approaches to C-lignin engineering in Medicago truncatula.

BACKGROUND: C-lignin is a homopolymer of caffeyl alcohol present in the seed coats of a variety of plant species including vanilla orchid, various cacti, and the ornamental plant Cleome hassleriana. Because of its unique chemical and physical properties, there is considerable interest in engineering C-lignin into the cell walls of bioenergy crops as a high-value co-product of bioprocessing. We have used information from a transcriptomic analysis of developing C. hassleriana seed coats to suggest strategies for engineering C-lignin in a heterologous system, using hairy roots of the model legume Medicago truncatula. RESULTS: We systematically tested strategies for C-lignin engineering using a combination of gene overexpression and RNAi-mediated knockdown in the caffeic acid/5-hydroxy coniferaldehyde 3/5-O-methyltransferase (comt) mutant background, monitoring the outcomes by analysis of lignin composition and profiling of monolignol pathway metabolites. In all cases, C-lignin accumulation required strong down-regulation of caffeoyl CoA 3-O-methyltransferase (CCoAOMT) paired with loss of function of COMT. Overexpression of the Selaginella moellendorffii ferulate 5-hydroxylase (SmF5H) gene in comt mutant hairy roots resulted in lines that unexpectedly accumulated high levels of S-lignin. CONCLUSION: C-Lignin accumulation of up to 15% of total lignin in lines with the greatest reduction in CCoAOMT expression required the strong down-regulation of both COMT and CCoAOMT, but did not require expression of a heterologous laccase, cinnamyl alcohol dehydrogenase (CAD) or cinnamoyl CoA reductase (CCR) with preference for 3,4-dihydroxy-substituted substrates in M. truncatula hairy roots. Cell wall fractionation studies suggested that the engineered C-units are not present in a heteropolymer with the bulk of the G-lignin.

Growth-defense trade-offs and yield loss in plants with engineered cell walls.

As a major component of plant secondary cell walls, lignin provides structural integrity and rigidity, and contributes to primary defense by providing a physical barrier to pathogen ingress. Genetic modification of lignin biosynthesis has been adopted to reduce the recalcitrance of lignified cell walls to improve biofuel production, tree pulping properties and forage digestibility. However, lignin-modification is often, but unpredictably, associated with dwarf phenotypes. Hypotheses suggested to explain this include: collapsed vessels leading to defects in water and solute transport; accumulation of molecule(s) that are inhibitory to plant growth or deficiency of metabolites that are critical for plant growth; activation of defense pathways linked to cell wall integrity sensing. However, there is still no commonly accepted underlying mechanism for the growth defects. Here, we discuss recent data on transcriptional reprogramming in plants with modified lignin content and their corresponding suppressor mutants, and evaluate growth-defense trade-offs as a factor underlying the growth phenotypes. New approaches will be necessary to estimate how gross changes in transcriptional reprogramming may quantitatively affect growth. Better understanding of the basis for yield drag following cell wall engineering is important for the biotechnological exploitation of plants as factories for fuels and chemicals.

Functional Genomics in the Study of Metabolic Pathways in Medicago truncatula: An Overview.

In addition to its value as a model system for studies on symbiotic nitrogen fixation, Medicago truncatula has recently become an organism of choice for dissection of complex pathways of secondary metabolism. This work has been driven by two main reasons, both with practical implications. First Medicago species possess a wide range of flavonoid and terpenoid natural products, many of which, for example, the isoflavonoids and triterpene saponins, have important biological activities impacting both plant and animal (including human) health. Second, M. truncatula serves as an excellent model for alfalfa, the world's major forage legume, and forage quality is determined in large part by the concentrations of products of secondary metabolism, particularly lignin and condensed tannins. We here review recent progress in understanding the pathways leading to flavonoids, lignin, and triterpene saponins through utilization of genetic resources in M. truncatula.

An essential role of caffeoyl shikimate esterase in monolignol biosynthesis in Medicago truncatula.

Biochemical and genetic analyses have previously identified caffeoyl shikimate esterase (CSE) as an enzyme in the monolignol biosynthesis pathway in Arabidopsis thaliana, although the generality of this finding has been questioned. Here we show the presence of CSE genes and associated enzyme activity in barrel medic (Medicago truncatula, dicot, Leguminosae), poplar (Populus deltoides, dicot, Salicaceae), and switchgrass (Panicum virgatum, monocot, Poaceae). Loss of function of CSE in transposon insertion lines of M. truncatula results in severe dwarfing, altered development, reduction in lignin content, and preferential accumulation of hydroxyphenyl units in lignin, indicating that the CSE enzyme is critical for normal lignification in this species. However, the model grass Brachypodium distachyon and corn (Zea mays) do not possess orthologs of the currently characterized CSE genes, and crude protein extracts from stems of these species exhibit only a weak esterase activity with caffeoyl shikimate. Our results suggest that the reaction catalyzed by CSE may not be essential for lignification in all plant species.

Comprehensive analysis of CLE polypeptide signaling gene expression and overexpression activity in Arabidopsis.

Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis (Arabidopsis thaliana), only a few families of extracellular signaling molecules have been discovered, and their biological roles are largely unknown. To expand our insight into the developmental processes potentially regulated by ligand-mediated signal transduction pathways, we undertook a systematic expression analysis of the members of the Arabidopsis CLAVATA3/ESR-RELATED (CLE) small signaling polypeptide family. Using reporter constructs, we show that the CLE genes have distinct and specific patterns of promoter activity. We find that each Arabidopsis tissue expresses at least one CLE gene, indicating that CLE-mediated signaling pathways are likely to play roles in many biological processes during the plant life cycle. Some CLE genes that are closely related in sequence have dissimilar expression profiles, yet in many tissues multiple CLE genes have overlapping patterns of promoter-driven reporter activity. This observation, plus the general absence of detectable morphological phenotypes in cle null mutants, suggest that a high degree of functional redundancy exists among CLE gene family members. Our work establishes a community resource of CLE-related biological materials and provides a platform for understanding and ultimately manipulating many different plant signaling systems.

Shoot apical meristem form and function.

The shoot apical meristem (SAM) generates above-ground aerial organs throughout the lifespan of higher plants. In order to fulfill this function, the meristem must maintain a balance between the self-renewal of a reservoir of central stem cells and organ initiation from peripheral cells. The activity of the pluripotent stem cell population in the SAM is dynamically controlled by complex, overlapping signaling networks that include the feedback regulation of meristem maintenance genes and the signaling of plant hormones. Organ initiation likewise requires the function of multifactor gene regulatory networks, as well as instructive cues from the plant hormone auxin and reciprocal signals from the shoot meristem. Floral meristems (FMs) are products of the reproductive SAM that sustains a transient stem cell reservoir for flower formation. Regulation of FM activity involves both feedback loops shared with the SAM and floral-specific factors. Recent studies have rapidly advanced our understanding of SAM function by adopting newly developed molecular and computational techniques. These advances are becoming integrated with data from traditional molecular genetics methodologies to develop a framework for understanding the central principles of SAM function.

Analyzing shoot apical meristem development.

The shoot apical meristem of Arabidopsis thaliana contains a reservoir of pluripotent stem cells that functions as a continuous source of new cells for organ formation during development. The SAM forms during embryogenesis, when it becomes stratified into specific cell layers and zones that can be delineated based on morphological and molecular criteria. The primary SAM produces all the aerial structures of the adult plant, and alterations in SAM organization or function can have profound effects on vegetative and reproductive plant morphology. Such SAM-specific defects can be identified, evaluated, and quantified using specialized microscopic and histological techniques.

Control of Arabidopsis leaf morphogenesis through regulation of the YABBY and KNOX families of transcription factors.

The patterning of initiating organs along specific axes of polarity is critical for the proper development of all higher organisms. Plant lateral organs, such as leaves, are derived from the shoot apical meristems located at the growing tips. After initiation, the leaf primordia of species such as Arabidopsis thaliana differentiate into a polarized structure consisting of a proximal petiole and a distal blade, but the molecular mechanisms that control proximal-distal pattern formation are poorly understood. The transcriptional activators BLADE-ON-PETIOLE1 (BOP1) and BOP2 are known to control Arabidopsis lateral organ differentiation by regulating gene expression along the adaxial-abaxial (dorsal-ventral) and proximal-distal polarity axes. Here, we demonstrate that the development of ectopic blade tissue along bop1 bop2 leaf petioles is strongly suppressed in a dosage-dependant manner by mutations in either of two closely related YABBY (YAB) genes, FILAMENTOUS FLOWER (FIL) and YAB3. Three KNOTTED-LIKE HOMEOBOX (KNOX1) genes also make lesser, and partially redundant, contributions to ectopic blade development in bop1 bop2 leaves. Mutation of these YAB and KNOX1 genes together causes nearly complete suppression of bop1 bop2 ectopic organ outgrowth at the morphological and cellular levels. Our data demonstrate that BOP1 and BOP2 regulate leaf patterning by controlling YAB and KNOX1 gene activity in the developing petiole.

BLADE-ON-PETIOLE1 coordinates organ determinacy and axial polarity in arabidopsis by directly activating ASYMMETRIC LEAVES2.

Continuous organ formation is a hallmark of plant development that requires organ-specific gene activity to establish determinacy and axial patterning, yet the molecular mechanisms that coordinate these events remain poorly understood. Here, we show that the organ-specific BTB-POZ domain proteins BLADE-ON-PETIOLE1 (BOP1) and BOP2 function as transcriptional activators during Arabidopsis thaliana leaf formation. We identify as a direct target of BOP1 induction the ASYMMETRIC LEAVES2 (AS2) gene, which promotes leaf cell fate specification and adaxial polarity. We find that BOP1 associates with the AS2 promoter and that BOP1 and BOP2 are required for AS2 activation specifically in the proximal, adaxial region of the leaf, demonstrating a role for the BOP proteins as proximal-distal as well as adaxial-abaxial patterning determinants. Furthermore, repression of BOP1 and BOP2 expression by the indeterminacy-promoting KNOX gene SHOOTMERISTEMLESS is critical to establish a functional embryonic shoot apical meristem. Our data indicate that direct activation of AS2 transcription by BOP1 and BOP2 is vital for generating the conditions for KNOX repression at the leaf base and may represent a conserved mechanism for coordinating leaf morphogenesis with patterning along the adaxial-abaxial and the proximal-distal axes.

BLADE-ON-PETIOLE 1 and 2 control Arabidopsis lateral organ fate through regulation of LOB domain and adaxial-abaxial polarity genes.

We report a novel function for BLADE-ON-PETIOLE1 (BOP1) and BOP2 in regulating Arabidopsis thaliana lateral organ cell fate and polarity, through the analysis of loss-of-function mutants and transgenic plants that ectopically express BOP1 or BOP2. 35S:BOP1 and 35S:BOP2 plants exhibit a very short and compact stature, hyponastic leaves, and downward-orienting siliques. We show that the LATERAL ORGAN BOUNDARIES (LOB) domain genes ASYMMETRIC LEAVES2 (AS2) and LOB are upregulated in 35S:BOP and downregulated in bop mutant plants. Ectopic expression of BOP1 or BOP2 also results in repression of class I knox gene expression. We further demonstrate a role for BOP1 and BOP2 in establishing the adaxial-abaxial polarity axis in the leaf petiole, where they regulate PHB and FIL expression and overlap in function with AS1 and AS2. Interestingly, during this study, we found that KANADI1 (KAN1) and KAN2 act to promote adaxial organ identity in addition to their well-known role in promoting abaxial organ identity. Our data indicate that BOP1 and BOP2 act in cells adjacent to the lateral organ boundary to repress genes that confer meristem cell fate and induce genes that promote lateral organ fate and polarity, thereby restricting the developmental potential of the organ-forming cells and facilitating their differentiation.

BLADE-ON-PETIOLE1 encodes a BTB/POZ domain protein required for leaf morphogenesis in Arabidopsis thaliana.

The BLADE-ON-PETIOLE1 (BOP1) gene of Arabidopsis thaliana is required for proper leaf morphogenesis. BOP1 regulates leaf differentiation in a proximal-distal manner, and represses the expression of three class I knotted-like homeobox (knox) genes during leaf formation. Utilizing a map-based approach, we identified the molecular nature of the BOP1 gene, which encodes a BTB/POZ domain protein with ankyrin repeats. BOP1 is a member of a small gene family in Arabidopsis that includes the disease resistance regulatory protein NPR1. Insertions in and around BOP1 cause distinct lesions in leaf morphogenesis, revealing complex regulation of the locus. BOP1 transcripts are initially detectable in embryos, where they specifically localize to the base of the developing cotyledons near the SAM. During vegetative development, BOP1 is expressed in young leaf primordia and at the base of the rosette leaves on the adaxial side. During reproductive development, BOP1 transcripts are detected in young floral buds, and at the base of the sepals and petals. Our results indicate that BOP1 encodes a putative regulatory protein that modulates meristematic activity at discrete locations in developing lateral organs. This is the first report on a plant protein that plays a key role in morphogenesis with the distinctive combinatorial architecture of the BTB/POZ and ankyrin repeat domains.

The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis.

The plant leaf provides an ideal system to study the mechanisms of organ formation and morphogenesis. The key factors that control leaf morphogenesis include the timing, location and extent of meristematic activity during cell division and differentiation. We identified an Arabidopsis mutant in which the regulation of meristematic activities in leaves was aberrant. The recessive mutant allele blade-on-petiole1-1 (bop1-1) produced ectopic, lobed blades along the adaxial side of petioles of the cotyledon and rosette leaves. The ectopic organ, which has some of the characteristics of rosette leaf blades with formation of trichomes in a dorsoventrally dependent manner, was generated by prolonged and clustered cell division in the mutant petioles. Ectopic, lobed blades were also formed on the proximal part of cauline leaves that lacked a petiole. Thus, BOP1 regulates the meristematic activity of leaf cells in a proximodistally dependent manner. Manifestation of the phenotypes in the mutant leaves was dependent on the leaf position. Thus, BOP1 controls leaf morphogenesis through control of the ectopic meristematic activity but within the context of the leaf proximodistality, dorsoventrality and heteroblasty. BOP1 appears to regulate meristematic activity in organs other than leaves, since the mutation also causes some ectopic outgrowths on stem surfaces and at the base of floral organs. Three class I knox genes, i.e., KNAT1, KNAT2 and KNAT6, were expressed aberrantly in the leaves of the bop1-1 mutant. Furthermore, the bop1-1 mutation showed some synergistic effect in double mutants with as1-1 or as2-2 mutation that is known to be defective in the regulation of meristematic activity and class I knox gene expression in leaves. The bop1-1 mutation also showed a synergistic effect with the stm-1 mutation, a strong mutant allele of a class I knox gene, STM. We, thus, suggest that BOP1 promotes or maintains a developmentally determinate state in leaf cells through the regulation of class I knox genes.

Involvement of the VEP1 gene in vascular strand development in Arabidopsis thaliana.

A dominant mutant line characterized by abnormal leaf venation pattern was isolated from a transgenic Arabidopsis plant pool that was generated with Agrobacterium culture harboring an Arabidopsis antisense cDNA library. In the mutant line, the phenotype was due to antisense suppression of a gene we named VEP1 (Vein Patterning). The predicted amino acid sequence of the gene contained a motif related to the mammalian death domain that is found in the apoptotic machinery. Reduced expression of the VEP1 gene resulted in the reduced complexity of the venation pattern of the cotyledons and foliar leaves, which was mainly due to the reduced number of the minor veins and their incomplete connection. The analysis of mutant embryos indicated that the phenotype was originated, at least in part, from a defect in the procambium patterning. In the mutant, the stem and root were thinner than those in wild type. This phenotype was associated with reduced vascular development. The promoter activity of the VEP1 gene was detected preferentially in the vascular regions. We propose that the death domain-containing protein VEP1 functions as a positive element required for vascular strand development in Arabidopsis thaliana.

Random antisense cDNA mutagenesis as an efficient functional genomic approach in higher plants.

Most cellular processes in an organism depend on functions of expressed sequences. Thus, efficient large-scale functional assignment of expressed sequences is crucial for understanding cellular processes. Towards this goal in plants, we designed a "random antisense cDNA mutagenesis (RAM)" approach. In a pilot experiment, 1,000 transgenic plants of Arabidopsis thaliana (L.) Heynh. (ecotype Wassilevskija) expressing random antisense cDNA(s) were generated from Agrobacterium cultures harboring an Arabidopsis antisense cDNA library. We identified 104 mutant lines from the transgenic pool by visual screening. Genetic analysis suggested that 37% of the mutations were likely due to antisense effects. When the cDNA inserts were isolated from 11 mutant lines by polymerase chain reaction and reintroduced into plants to express the antisense transcripts, the original mutant phenotypes were reproduced in 7 cDNA clones. One of the cDNA clones did not generate a database match to any sequence with known functions, but did have a dramatic effect on the architecture of the inflorescence in the antisense transgenic plants. Through the RAM approach, it should be possible to assign a large number of expressed sequences to known in vivo functions in plants.