Molecular Advances of Bud Dormancy in Trees.

Seasonal bud dormancy in perennial woody plants is a crucial and intricate process that is vital for the survival and development of plants. Over the past few decades, significant advancements have been made in understanding many features of bud dormancy, particularly in model species, where certain molecular mechanisms underlying this process have been elucidated. In this review, we provide an overview of recent molecular progress in understanding bud dormancy in trees, with a specific emphasis on the integration of common signaling and molecular mechanisms identified across different tree species. Additionally, we address some challenges that have emerged in the in-depth understanding of bud dormancy and offer insights for future studies.

EARLY BUD-BREAK 1 and EARLY BUD-BREAK 3 control resumption of poplar growth after winter dormancy.

Bud-break is an economically and environmentally important process in trees and shrubs from boreal and temperate latitudes, but its molecular mechanisms are poorly understood. Here, we show that two previously reported transcription factors, EARLY BUD BREAK 1 (EBB1) and SHORT VEGETATIVE PHASE-Like (SVL) directly interact to control bud-break. EBB1 is a positive regulator of bud-break, whereas SVL is a negative regulator of bud-break. EBB1 directly and negatively regulates SVL expression. We further report the identification and characterization of the EBB3 gene. EBB3 is a temperature-responsive, epigenetically-regulated, positive regulator of bud-break that provides a direct link to activation of the cell cycle during bud-break. EBB3 is an AP2/ERF transcription factor that positively and directly regulates CYCLIND3.1 gene. Our results reveal the architecture of a putative regulatory module that links temperature-mediated control of bud-break with activation of cell cycle.

Plant Development: Dual Roles of Poplar SVL in Vegetative Bud Dormancy.

Vegetative bud dormancy is an important adaptive process allowing survival over winter in trees from temperate and boreal regions. A recent discovery implicates a MADS-box transcription factor from poplar in regulation of both entry and release from dormancy.

Manipulation of Growth and Architectural Characteristics in Trees for Increased Woody Biomass Production.

Growth and architectural traits in trees are economically and environmentally important and thus of considerable importance to the improvement of forest and fruit trees. These traits are complex and result from the operation of a number of molecular mechanisms. This review will focus on the regulation of crown architecture, secondary woody growth and adventitious rooting. These traits and processes have significant impact on deployment, management, and productivity of tree crops. The majority of the described work comes from experiments in model plants, poplar, apple, peach, and plum because these species allow functional analysis of the involved genes and have significant genomics resources. However, these studies convincingly show conserved mechanisms for elaboration of specific growth and architectural traits. The conservation of these mechanisms suggest that they can be used as a blueprint for the improvement of these traits and processes in phylogenetically diverse tree crops. We will specifically consider the involvement of flowering time, transcription factors and hormone-associated genes. The review will also discuss the impact of recent technological advances as well as the challenges to the dissection of these traits in trees.

BIG LEAF is a regulator of organ size and adventitious root formation in poplar.

Here we report the discovery through activation tagging and subsequent characterization of the BIG LEAF (BL) gene from poplar. In poplar, BL regulates leaf size via positively affecting cell proliferation. Up and downregulation of the gene led to increased and decreased leaf size, respectively, and these phenotypes corresponded to increased and decreased cell numbers. BL function encompasses the early stages of leaf development as native BL expression was specific to the shoot apical meristem and leaf primordia and was absent from the later stages of leaf development and other organs. Consistently, BL downregulation reduced leaf size at the earliest stages of leaf development. Ectopic expression in mature leaves resulted in continued growth most probably via sustained cell proliferation and thus the increased leaf size. In contrast to the positive effect on leaf growth, ectopic BL expression in stems interfered with and significantly reduced stem thickening, suggesting that BL is a highly specific activator of growth. In addition, stem cuttings from BL overexpressing plants developed roots, whereas the wild type was difficult to root, demonstrating that BL is a positive regulator of adventitious rooting. Large transcriptomic changes in plants that overexpressed BL indicated that BL may have a broad integrative role, encompassing many genes linked to organ growth. We conclude that BL plays a fundamental role in control of leaf size and thus may be a useful tool for modifying plant biomass productivity and adventitious rooting.

EARLY BUD-BREAK 1 (EBB1) is a regulator of release from seasonal dormancy in poplar trees.

Trees from temperate latitudes transition between growth and dormancy to survive dehydration and freezing stress during winter months. We used activation tagging to isolate a dominant mutation affecting release from dormancy and identified the corresponding gene EARLY BUD-BREAK 1 (EBB1). We demonstrate through positioning of the tag, expression analysis, and retransformation experiments that EBB1 encodes a putative APETALA2/Ethylene responsive factor transcription factor. Transgenic up-regulation of the gene caused early bud-flush, whereas down-regulation delayed bud-break. Native EBB1 expression was highest in actively growing apices, undetectable during the dormancy period, but rapidly increased before bud-break. The EBB1 transcript was localized in the L1/L2 layers of the shoot meristem and leaf primordia. EBB1-overexpressing transgenic plants displayed enlarged shoot meristems, open and poorly differentiated buds, and a higher rate of cell division in the apex. Transcriptome analyses of the EBB1 transgenics identified 971 differentially expressed genes whose expression correlated with the EBB1 expression changes in the transgenic plants. Promoter analysis among the differentially expressed genes for the presence of a canonical EBB1-binding site identified 65 putative target genes, indicative of a broad regulatory context of EBB1 function. Our results suggest that EBB1 has a major and integrative role in reactivation of meristem activity after winter dormancy.

Roles of gibberellin catabolism and signaling in growth and physiological response to drought and short-day photoperiods in Populus trees.

Survival and productivity of perennial plants in temperate zones are dependent on robust responses to prolonged and seasonal cycles of unfavorable conditions. Here we report whole-genome microarray, expression, physiological, and transgenic evidence in hybrid poplar (Populus tremula × Populus alba) showing that gibberellin (GA) catabolism and repressive signaling mediates shoot growth inhibition and physiological adaptation in response to drought and short-day (SD) induced bud dormancy. Both water deprivation and SDs elicited activation of a suite of poplar GA2ox and DELLA encoding genes. Poplar transgenics with up-regulated GA 2-oxidase (GA2ox) and DELLA domain proteins showed hypersensitive growth inhibition in response to both drought and SDs. In addition, the transgenic plants displayed greater drought resistance as evidenced by increased pigment concentrations (chlorophyll and carotenoid) and reductions in electrolyte leakage (EL). Comparative transcriptome analysis using whole-genome microarray showed that the GA-deficiency and GA-insensitivity, SD-induced dormancy, and drought response in poplar share a common regulon of 684 differentially-expressed genes, which suggest GA metabolism and signaling plays a role in plant physiological adaptations in response to alterations in environmental factors. Our results demonstrate that GA catabolism and repressive signaling represents a major route for control of growth and physiological adaptation in response to immediate or imminent adverse conditions.

DR5 as a reporter system to study auxin response in Populus.

KEY MESSAGE : Auxin responsive promoter DR5 reporter system is functional in Populus to monitor auxin response in tissues including leaves, roots, and stems. We described the behavior of the DR5::GUS reporter system in stably transformed Populus plants. We found several similarities with Arabidopsis, including sensitivity to native and synthetic auxins, rapid induction after treatment in a variety of tissues, and maximal responses in root tissues. There were also several important differences from Arabidopsis, including slower time to maximum response and lower induction amplitude. Young leaves and stem sections below the apex showed much higher DR5 activity than did older leaves and stems undergoing secondary growth. DR5 activity was highest in cortex, suggesting high levels of auxin concentration and/or sensitivity in this tissue. Our study shows that the DR5 reporter system is a sensitive and facile system for monitoring auxin responses and distribution at cellular resolution in poplar.

The AINTEGUMENTA LIKE1 homeotic transcription factor PtAIL1 controls the formation of adventitious root primordia in poplar.

Adventitious rooting is an essential but sometimes rate-limiting step in the clonal multiplication of elite tree germplasm, because the ability to form roots declines rapidly with age in mature adult plant tissues. In spite of the importance of adventitious rooting, the mechanism behind this developmental process remains poorly understood. We have described the transcriptional profiles that are associated with the developmental stages of adventitious root formation in the model tree poplar (Populus trichocarpa). Transcriptome analyses indicate a highly specific temporal induction of the AINTEGUMENTA LIKE1 (PtAIL1) transcription factor of the AP2 family during adventitious root formation. Transgenic poplar samples that overexpressed PtAIL1 were able to grow an increased number of adventitious roots, whereas RNA interference mediated the down-expression of PtAIL1 expression, which led to a delay in adventitious root formation. Microarray analysis showed that the expression of 15 genes, including the transcription factors AGAMOUS-Like6 and MYB36, was overexpressed in the stem tissues that generated root primordia in PtAIL1-overexpressing plants, whereas their expression was reduced in the RNA interference lines. These results demonstrate that PtAIL1 is a positive regulator of poplar rooting that acts early in the development of adventitious roots.

PHOTOPERIOD RESPONSE 1 (PHOR1)-like genes regulate shoot/root growth, starch accumulation, and wood formation in Populus.

This study describes functional characterization of two putative poplar PHOTOPERIOD RESPONSE 1 (PHOR1) orthologues. The expression and sequence analyses indicate that the two poplar genes diverged, at least partially, in function. PtPHOR1_1 is most highly expressed in roots and induced by short days, while PtPHOR1_2 is more uniformly expressed throughout plant tissues and is not responsive to short days. The two PHOR1 genes also had distinct effects on shoot and root growth when their expression was up- and downregulated transgenically. PtPHOR1_1 effects were restricted to roots while PtPHOR1_2 had similar effects on aerial and below-ground development. Nevertheless, both genes seemed to be upregulated in transgenic poplars that are gibberellin-deficient and gibberellin-insensitive, suggesting interplay with gibberellin signalling. PHOR1 suppression led to increased starch accumulation in both roots and stems. The effect of PHOR1 suppression on starch accumulation was coupled with growth-inhibiting effects in both roots and shoots, suggesting that PHOR1 is part of a mechanism that regulates the allocation of carbohydrate to growth or storage in poplar. PHOR1 downregulation led to significant reduction of xylem formation caused by smaller fibres and vessels suggesting that PHOR1 likely plays a role in the growth of xylem cells.

Green revolution trees: semidwarfism transgenes modify gibberellins, promote root growth, enhance morphological diversity, and reduce competitiveness in hybrid poplar.

Semidwarfism has been used extensively in row crops and horticulture to promote yield, reduce lodging, and improve harvest index, and it might have similar benefits for trees for short-rotation forestry or energy plantations, reclamation, phytoremediation, or other applications. We studied the effects of the dominant semidwarfism transgenes GA Insensitive (GAI) and Repressor of GAI-Like, which affect gibberellin (GA) action, and the GA catabolic gene, GA 2-oxidase, in nursery beds and in 2-year-old high-density stands of hybrid poplar (Populus tremula × Populus alba). Twenty-nine traits were analyzed, including measures of growth, morphology, and physiology. Endogenous GA levels were modified in most transgenic events; GA(20) and GA(8), in particular, had strong inverse associations with tree height. Nearly all measured traits varied significantly among genotypes, and several traits interacted with planting density, including aboveground biomass, root-shoot ratio, root fraction, branch angle, and crown depth. Semidwarfism promoted biomass allocation to roots over shoots and substantially increased rooting efficiency with most genes tested. The increased root proportion and increased leaf chlorophyll levels were associated with changes in leaf carbon isotope discrimination, indicating altered water use efficiency. Semidwarf trees had dramatically reduced growth when in direct competition with wild-type trees, supporting the hypothesis that semidwarfism genes could be effective tools to mitigate the spread of exotic, hybrid, and transgenic plants in wild and feral populations.

SHORT INTERNODES-like genes regulate shoot growth and xylem proliferation in Populus.

• Genes controlling plant growth and form are of considerable interest, because they affect survival and productivity traits, and are largely unknown or poorly characterized. The SHORT INTERNODES(SHI) gene is one of a 10-member SHI-RELATED SEQUENCE (SRS) gene family in Arabidopsis that includes important developmental regulators. • Using comparative sequence analysis of the SRS gene families in poplar and Arabidopsis, we identified two poplar proteins that are most similar to SHI and its closely related gene STYLISH1 (STY1). The two poplar genes are very similar in sequence and expression and are therefore probably paralogs with redundant functions. • RNAi suppression of the two Populus genes enhanced shoot and root growth, whereas the overexpression of Arabidopsis SHI in poplar reduced internode and petiole length. The suppression of the two genes increased fiber length and the proportion of xylem tissue, mainly through increased xylem cell proliferation. The transgenic modifications were also associated with significant changes in the concentrations of gibberellins and cytokinin. • We conclude that Populus SHI-RELATED SEQUENCE (SRS) genes play an important role in the regulation of vegetative growth, including wood formation, and thus could be useful tools for the modification of biomass productivity, wood quality or plant form.

Gibberellins regulate lateral root formation in Populus through interactions with auxin and other hormones.

The role of gibberellins (GAs) in regulation of lateral root development is poorly understood. We show that GA-deficient (35S:PcGA2ox1) and GA-insensitive (35S:rgl1) transgenic Populus exhibited increased lateral root proliferation and elongation under in vitro and greenhouse conditions, and these effects were reversed by exogenous GA treatment. In addition, RNA interference suppression of two poplar GA 2-oxidases predominantly expressed in roots also decreased lateral root formation. GAs negatively affected lateral root formation by inhibiting lateral root primordium initiation. A whole-genome microarray analysis of root development in GA-modified transgenic plants revealed 2069 genes with significantly altered expression. The expression of 1178 genes, including genes that promote cell proliferation, growth, and cell wall loosening, corresponded to the phenotypic severity of the root traits when transgenic events with differential phenotypic expression were compared. The array data and direct hormone measurements suggested crosstalk of GA signaling with other hormone pathways, including auxin and abscisic acid. Transgenic modification of a differentially expressed gene encoding an auxin efflux carrier suggests that GA modulation of lateral root development is at least partly imparted by polar auxin transport modification. These results suggest a mechanism for GA-regulated modulation of lateral root proliferation associated with regulation of plant allometry during the stress response.

Genes for control of plant stature and form.

Here we summarize progress in identification of three classes of genes useful for control of plant architecture: those affecting hormone metabolism and signaling; transcription and other regulatory factors; and the cell cycle. We focus on strong modifiers of stature and form that may be useful for directed modification of plant architecture, rather than the detailed mechanisms of gene action. Gibberellin (GA) metabolic and response genes are particularly attractive targets for manipulation because many act in a dose-dependent manner; similar phenotypic effects can be readily achieved in heterologous species; and induced pleiotropic effects--such as on nitrogen assimilation, photosynthesis, and lateral root production--are usually positive with respect to crop performance. Genes encoding transcription factors represent strong candidates for manipulation of plant architecture. For example, AINTEGUMENTA, ARGOS (auxin-regulated gene controlling organ size), and growth-regulating factors (GRFs) are strong modifiers of leaf and/or flower size. Plants overexpressing these genes had increased organ size and did not display negative pleiotropic effects in glasshouse environments. TCP-domain genes such as CINCINNATA, and the associated regulatory miRNAs such as miRJAW, may provide useful means to modulate leaf curvature and other foliage properties. There are considerable opportunities for comparative and translational genomics in nonmodel plant systems.

Genetic transformation: a powerful tool for dissection of adaptive traits in trees.

Plant transformation and regeneration systems have become indispensable parts of gene discovery and functional characterization over the last two decades. Adoption of transformation methods in studies of plant adaptation to natural environments has been slow. This is a result of poor genomic knowledge and inefficient transformation systems for species dominating terrestrial ecosystems, and logistical difficulties in conducting field tests of genetically engineered organisms. In trees, where long generation cycles, high background polymorphism, large sizes and outcrossing systems of mating make production of near-isogenic lines and large experiments difficult, transformation is an attractive alternative for establishing direct linkages between genes and adaptively significant phenotypes. Here, we outline the capabilities, challenges, and prospects for transformation to become a significant tool for studying the ecophysiological adaptation of trees to the environment. Focusing on poplars (genus Populus) as model system, we describe how transformation-based approaches can provide insights into the genes that control adaptive traits. The availability of the poplar genome sequence, along with its large expressed sequences tag (EST) databanks, facile transformation and rapid growth, enable reverse genetic approaches to be used to test virtually any hypothesis of gene function.

An auxin-inducible gene from loblolly pine (Pinus taeda L.) is differentially expressed in mature and juvenile-phase shoots and encodes a putative transmembrane protein.

We have isolated a gene from loblolly pine, 5NG4, that is highly and specifically induced by auxin in juvenile loblolly pine shoots prior to adventitious root formation, but substantially down-regulated in physiologically mature shoots that are adventitious rooting incompetent. 5NG4 was highly auxin-induced in roots, stems and hypocotyls, organs that can form either lateral or adventitious roots following an auxin treatment, but was not induced to the same level in needles and cotyledons, organs that do not form roots. The deduced amino acid sequence shows homology to the MtN21 nodulin gene from Medicago truncatula. The expression pattern of 5NG4 and its homology to a protein from Medicago involved in a root-related process suggest a possible role for this gene in adventitious root formation. Homology searches also identified similar proteins in Arabidopsis thaliana and Oryza sativa. High conservation across these evolutionarily distant species suggests essential functions in plant growth and development. A 38-member family of genes homologous to 5NG4 was identified in the A. thaliana genome. The physiological significance of this redundancy is most likely associated with functional divergence and/or expression specificity of the different family members. The exact biochemical function of the gene is still unknown, but sequence and structure predictions and 5NG4::GFP fusion protein localizations indicate it is a transmembrane protein with a possible transport function.

Poplar genome sequence: functional genomics in an ecologically dominant plant species.

In addition to their value for wood products, members of the genus Populus (poplars) provide a range of ecological services, including carbon sequestration, bioremediation, nutrient cycling, biofiltration and diverse habitats. They are also widely used model organisms for tree molecular biology and biotechnology. The sequencing of the poplar genome to an approximately 6x depth adds to a long list of important attributes for research. These include facile transformation, vegetative propagation, rapid growth, modest genome size and extensive expressed sequence tags. Here, we discuss how the genome sequence and transformability of poplar, together with its high levels of genetic and ecological diversity, are enabling new insights into the genetic programs controlling ontogeny, ecological adaptation and environmental physiology of trees.

Activation tagging of a dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that regulates tree stature.

We identified a dwarf transgenic hybrid poplar (Populus tremula x Populus alba) after screening of 627 independent activation-tagged transgenic lines in tissue culture, greenhouse, and field environments. The cause of the phenotype was a hyperactivated gene encoding GA 2-oxidase (GA2ox), the major gibberellin (GA) catabolic enzyme in plants. The mutation resulted from insertion of a strong transcriptional enhancer near the transcription start site. Overexpression of the poplar GA2ox gene (PtaGA2ox1) caused hyperaccumulation of mRNA transcripts, quantitative shifts in the spectrum of GAs, and similarity in phenotype to transgenic poplars that overexpress a bean (Phaseolus coccineus) GA2ox gene. The poplar PtaGA2ox1 sequence was most closely related to PsGA2ox2 from pea (Pisum sativum) and two poorly known GA2oxs from Arabidopsis (AtGA2ox4 and AtGA2ox5). The dwarf phenotype was reversible through gibberellic acid application to the shoot apex. Transgenic approaches to producing semidwarf trees for use in arboriculture, horticulture, and forestry could have significant economic and environmental benefits, including altered fiber and fruit production, greater ease of management, and reduced risk of spread in wild populations.