Activity-dormancy transition in the cambial meristem involves stage-specific modulation of auxin response in hybrid aspen.

The molecular basis of short-day-induced growth cessation and dormancy in the meristems of perennial plants (e.g., forest trees growing in temperate and high-latitude regions) is poorly understood. Using global transcript profiling, we show distinct stage-specific alterations in auxin responsiveness of the transcriptome in the stem tissues during short-day-induced growth cessation and both the transition to and establishment of dormancy in the cambial meristem of hybrid aspen trees. This stage-specific modulation of auxin signaling appears to be controlled via distinct mechanisms. Whereas the induction of growth cessation in the cambium could involve induction of repressor auxin response factors (ARFs) and down-regulation of activator ARFs, dormancy is associated with perturbation of the activity of the SKP-Cullin-F-box(TIR) (SCF(TIR)) complex, leading to potential stabilization of repressor auxin (AUX)/indole-3-acetic acid (IAA) proteins. Although the role of hormones, such as abscisic acid (ABA) and gibberellic acid (GA), in growth cessation and dormancy is well established, our data now implicate auxin in this process. Importantly, in contrast to most developmental processes in which regulation by auxin involves changes in cellular auxin contents, day-length-regulated induction of cambial growth cessation and dormancy involves changes in auxin responses rather than auxin content.

Components acting downstream of short day perception regulate differential cessation of cambial activity and associated responses in early and late clones of hybrid poplar.

Short days (SDs) in autumn induce growth cessation, bud set, cold acclimation, and dormancy in trees of boreal and temperate forests, and these responses occur earlier in northern than in southern genotypes. Nevertheless, we know little about whether this variation results from differential perception of SDs or differential downstream responses to the SD signal or a combination of the two. We compared global patterns of SD-regulated gene expression in the stems of hybrid poplar (Populus trichocarpa × Populus deltoides) clones that differ in their SD-induced growth cessation in order to address this question. The timing of cessation of cambial cell division caused by SDs differed between the clones and was coincident with the change in the pattern of expression of the auxin-regulated genes. The clones also differed in the timing of their SD-regulated changes in the transcript abundance of genes associated with cold tolerance, starch breakdown, and storage protein accumulation. By analyzing the expression of homologs of FLOWERING LOCUS T, we demonstrated that the clones differed little in their perception of SDs under the growth conditions applied but differed substantially in the downstream responses manifested in the timing and magnitude of gene expression after SD treatment. These results demonstrate the existence of factors that act downstream of SD perception and can contribute to variation in SD-regulated adaptive photoperiodic responses in trees.

Role of the GNOM gene in Arabidopsis apical-basal patterning--From mutant phenotype to cellular mechanism of protein action.

How the apical-basal axis of polarity is established in embryogenesis is still a mystery in plant development. This axis appeared specifically compromised by mutations in the Arabidopsis GNOM gene. Surprisingly, GNOM encodes an ARF guanine-nucleotide exchange factor (ARF-GEF) that regulates the formation of vesicles in membrane trafficking. In-depth functional analysis of GNOM and its closest relative, GNOM-LIKE 1 (GNL1), has provided a mechanistic explanation for the development-specific role of a seemingly mundane trafficking regulator. The current model proposes that GNOM is specifically involved in the endosomal recycling of the auxin-efflux carrier PIN1 to the basal plasma membrane in provascular cells, which in turn is required for the accumulation of the plant hormone auxin at the future root pole through polar auxin transport. Thus, the analysis of GNOM highlights the importance of cell-biological processes for a mechanistic understanding of development.

Functional diversification of closely related ARF-GEFs in protein secretion and recycling.

Guanine-nucleotide exchange factors on ADP-ribosylation factor GTPases (ARF-GEFs) regulate vesicle formation in time and space by activating ARF substrates on distinct donor membranes. Mammalian GBF1 (ref. 2) and yeast Gea1/2 (ref. 3) ARF-GEFs act at Golgi membranes, regulating COPI-coated vesicle formation. In contrast, their Arabidopsis thaliana homologue GNOM (GN) is required for endosomal recycling, playing an important part in development. This difference indicates an evolutionary divergence of trafficking pathways between animals and plants, and raised the question of how endoplasmic reticulum-Golgi transport is regulated in plants. Here we demonstrate that the closest homologue of GNOM in Arabidopsis, GNOM-LIKE1 (GNL1; NM_123312; At5g39500), performs this ancestral function. GNL1 localizes to and acts primarily at Golgi stacks, regulating COPI-coated vesicle formation. Surprisingly, GNOM can functionally substitute for GNL1, but not vice versa. Our results suggest that large ARF-GEFs of the GBF1 class perform a conserved role in endoplasmic reticulum-Golgi trafficking and secretion, which is done by GNL1 and GNOM in Arabidopsis, whereas GNOM has evolved to perform an additional plant-specific function of recycling from endosomes to the plasma membrane. Duplication and diversification of ARF-GEFs in plants contrasts with the evolution of entirely new classes of ARF-GEFs for endosomal trafficking in animals, which illustrates the independent evolution of complex endosomal pathways in the two kingdoms.

Environmental and hormonal regulation of the activity-dormancy cycle in the cambial meristem involves stage-specific modulation of transcriptional and metabolic networks.

We have performed transcript and metabolite profiling of isolated cambial meristem cells of the model tree aspen during the course of their activity-dormancy cycle to better understand the environmental and hormonal regulation of this process in perennial plants. Considerable modulation of cambial transcriptome and metabolome occurs throughout the activity-dormancy cycle. However, in addition to transcription, post-transcriptional control is also an important regulatory mechanism as exemplified by the regulation of cell-cycle genes during the reactivation of cambial cell division in the spring. Genes related to cold hardiness display temporally distinct induction patterns in the autumn which could explain the step-wise development of cold hardiness. Factors other than low temperature regulate the induction of early cold hardiness-related genes whereas abscisic acid (ABA) could potentially regulate the induction of late cold hardiness-related genes in the autumn. Starch breakdown in the autumn appears to be regulated by the 'short day' signal and plays a key role in providing substrates for the production of energy, fatty acids and cryoprotectants. Catabolism of sucrose and fats provides energy during the early stages of reactivation in the spring, whereas the reducing equivalents are generated through activation of the pentose phosphate shunt. Modulation of gibberellin (GA) signaling and biosynthesis could play a key role in the regulation of cambial activity during the activity-dormancy cycle as suggested by the induction of PttRGA which encodes a negative regulator of growth in the autumn and that of a GA-20 oxidase, a key gibberellin biosynthesis gene during reactivation in spring. In summary, our data reveal the dynamics of transcriptional and metabolic networks and identify potential targets of environmental and hormonal signals in the regulation of the activity-dormancy cycle in cambial meristem.

Carbohydrate-active enzymes involved in the secondary cell wall biogenesis in hybrid aspen.

Wood formation is a fundamental biological process with significant economic interest. While lignin biosynthesis is currently relatively well understood, the pathways leading to the synthesis of the key structural carbohydrates in wood fibers remain obscure. We have used a functional genomics approach to identify enzymes involved in carbohydrate biosynthesis and remodeling during xylem development in the hybrid aspen Populus tremula x tremuloides. Microarrays containing cDNA clones from different tissue-specific libraries were hybridized with probes obtained from narrow tissue sections prepared by cryosectioning of the developing xylem. Bioinformatic analyses using the sensitive tools developed for carbohydrate-active enzymes allowed the identification of 25 xylem-specific glycosyltransferases belonging to the Carbohydrate-Active EnZYme families GT2, GT8, GT14, GT31, GT43, GT47, and GT61 and nine glycosidases (or transglycosidases) belonging to the Carbohydrate-Active EnZYme families GH9, GH10, GH16, GH17, GH19, GH28, GH35, and GH51. While no genes encoding either polysaccharide lyases or carbohydrate esterases were found among the secondary wall-specific genes, one putative O-acetyltransferase was identified. These wood-specific enzyme genes constitute a valuable resource for future development of engineered fibers with improved performance in different applications.

MYB transcription factors are differentially expressed and regulated during secondary vascular tissue development in hybrid aspen.

More than 120,000 poplar ESTs have been sequenced from 20 different cDNA libraries by the Swedish Centre for Tree Functional Genomics. We screened this EST collection for MYB transcription factors involved in secondary vascular tissue formation, and genes assigned as PttMYB3Ra, PttMYB4a and PttMYB21a were selected for further characterisation. Three MYB genes showed different expression patterns in various organs, tissues and stem sub-sections representing different developmental stages of vascular tissue formation. Furthermore, the analysis showed that PttMYB21a expression was much higher in secondary cell wall formation zone of xylem and phloem fibers than in other developmental zones. Transgenic hybrid aspen plants, expressing the 3'-part of the PttMYB21a gene in antisense orientation were generated to assess the function of PttMYB21a gene in vascular tissue formation and lignification. All transgenic lines showed reduced growth and had fewer internodes compared to the wild-type. The analysis of selected lines showed that acid soluble lignin present in the bark was higher in transgenic lines as compared to wild-type plants. Moreover a higher transcript level of caffeoyl-CoA 3-O-methyltransferase [CCoAOMT]; EC 2.1.1.104) was found in the phloem of the transgenic plants, suggesting that PttMYB21a might function as a transcriptional repressor.

Cambial meristem dormancy in trees involves extensive remodelling of the transcriptome.

The establishment of the dormant state in meristems involves considerable physiological and metabolic alterations necessary for surviving unfavourable growth conditions. However, a global molecular analysis of dormancy in meristems has been hampered by the difficulty in isolating meristem cells. We used cryosectioning to isolate purified cambial meristem cells from the woody plant Populus tremula during active growth and dormancy. These samples were used to generate meristem-specific cDNA libraries and for cDNA microarray experiments to define the global transcriptional changes underlying cambial dormancy. The results indicate a significant reduction in the complexity of the cambial transcriptome in the dormant state. Although cell division is terminated in the dormant cambium, the cell cycle machinery appears to be maintained in a skeletal state as suggested by the continued presence of transcripts for several cell cycle regulators. The downregulation of PttPIN1 and PttPIN2 transcripts explains the reduced basipetal polar auxin transport during dormancy. The induction of a member of the SINA family of ubiquitin ligases implicated in auxin signalling indicates a potential mechanism for modulation of auxin sensitivity during cambial dormancy. The metabolic alterations during dormancy are mirrored in the induction of genes involved in starch breakdown and the glyoxysomal cycle. Interestingly, the induction of RGA1 like gene suggests modification of gibberellin signalling in cambial dormancy. The induction of genes such as poplar orthologues of FIE and HAP2 indicates a potential role for these global regulators of transcription in orchestrating extensive changes in gene expression during dormancy.

A high-resolution transcript profile across the wood-forming meristem of poplar identifies potential regulators of cambial stem cell identity.

Plant growth is the result of cell proliferation in meristems, which requires a careful balance between the formation of new tissue and the maintenance of a set of undifferentiated stem cells. Recent studies have provided important information on several genetic networks responsible for stem cell maintenance and regulation of cell differentiation in the apical meristems of shoots and roots. Nothing, however, is known about the regulatory networks in secondary meristems like the vascular cambium of trees. We have made use of the large size and highly regular layered organization of the cambial meristem to create a high-resolution transcriptional map covering 220 microm of the cambial region of aspen (Populus tremula). Clusters of differentially expressed genes revealed substantial differences in the transcriptomes of the six anatomically homogenous cell layers in the meristem zone. Based on transcriptional and anatomical data, we present a model for the position of the stem cells and the proliferating mother cells in the cambial zone. We also provide sets of marker genes for different stages of xylem and phloem differentiation and identify potential regulators of cambial meristem activity. Interestingly, analysis of known regulators of apical meristem development indicates substantial similarity in regulatory networks between primary and secondary meristems.

Expansins abundant in secondary xylem belong to subgroup A of the alpha-expansin gene family.

Differentiation of xylem cells in dicotyledonous plants involves expansion of the radial primary cell walls and intrusive tip growth of cambial derivative cells prior to the deposition of a thick secondary wall essential for xylem function. Expansins are cell wall-residing proteins that have an ability to plasticize the cellulose-hemicellulose network of primary walls. We found expansin activity in proteins extracted from the cambial region of mature stems in a model tree species hybrid aspen (Populus tremula x Populus tremuloides Michx). We identified three alpha-expansin genes (PttEXP1, PttEXP2, and PttEXP8) and one beta-expansin gene (PttEXPB1) in a cambial region expressed sequence tag library, among which PttEXP1 was most abundantly represented. Northern-blot analyses in aspen vegetative organs and tissues showed that PttEXP1 was specifically expressed in mature stems exhibiting secondary growth, where it was present in the cambium and in the radial expansion zone. By contrast, PttEXP2 was mostly expressed in developing leaves. In situ reverse transcription-PCR provided evidence for accumulation of mRNA of PttEXP1 along with ribosomal rRNA at the tips of intrusively growing xylem fibers, suggesting that PttEXP1 protein has a role in intrusive tip growth. An examination of tension wood and leaf cDNA libraries identified another expansin, PttEXP5, very similar to PttEXP1, as the major expansin in developing tension wood, while PttEXP3 was the major expansin expressed in developing leaves. Comparative analysis of expansins expressed in woody stems in aspen, Arabidopsis, and pine showed that the most abundantly expressed expansins share sequence similarities, belonging to the subfamily A of alpha-expansins and having two conserved motifs at the beginning and end of the mature protein, RIPVG and KNFRV, respectively. This conservation suggests that these genes may share a specialized, not yet identified function.

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen.

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.