The Chinese pine genome and methylome unveil key features of conifer evolution.

Conifers dominate the world's forest ecosystems and are the most widely planted tree species. Their giant and complex genomes present great challenges for assembling a complete reference genome for evolutionary and genomic studies. We present a 25.4-Gb chromosome-level assembly of Chinese pine (Pinus tabuliformis) and revealed that its genome size is mostly attributable to huge intergenic regions and long introns with high transposable element (TE) content. Large genes with long introns exhibited higher expressions levels. Despite a lack of recent whole-genome duplication, 91.2% of genes were duplicated through dispersed duplication, and expanded gene families are mainly related to stress responses, which may underpin conifers' adaptation, particularly in cold and/or arid conditions. The reproductive regulation network is distinct compared with angiosperms. Slow removal of TEs with high-level methylation may have contributed to genomic expansion. This study provides insights into conifer evolution and resources for advancing research on conifer adaptation and development.

The cytoskeleton controls the dynamics of plasma membrane proteins and facilitates their endocytosis in plants.

Plasma membranes (PMs) are highly dynamic structures where lipids and proteins can theoretically diffuse freely. However, reports indicate that PM proteins do not freely diffuse within their planes but are constrained by cytoskeleton networks, though the mechanisms for how the cytoskeleton restricts lateral diffusion of plant PM proteins are unclear. Through single-molecule tracking, we investigated the dynamics of six Arabidopsis (Arabidopsis thaliana) PM proteins with diverse structures and found distinctions in sizes and dynamics among these proteins. Moreover, we showed that the cytoskeleton, particularly microtubules, limits the diffusion of PM proteins, including transmembrane and membrane-anchoring proteins. Interestingly, the microfilament skeleton regulates intracellular transport of endocytic cargo. Therefore, these findings indicate that the cytoskeleton controls signal transduction by limiting diffusion of PM proteins in specific membrane compartments and participating in transport of internalized cargo vesicles, thus actively regulating plant signal transduction.

PagARGOS promotes low-lignin wood formation in poplar.

Wood formation, which occurs mainly through secondary xylem development, is important not only for supplying raw material for the 'ligno-chemical' industry but also for driving the storage of carbon. However, the complex mechanisms underlying the promotion of xylem formation remain to be elucidated. Here, we found that overexpression of Auxin-Regulated Gene involved in Organ Size (ARGOS) in hybrid poplar 84 K (Populus alba × Populus tremula var. glandulosa) enlarged organ size. In particular, PagARGOS promoted secondary growth of stems with increased xylem formation. To gain further insight into how PagARGOS regulates xylem development, we further carried out yeast two-hybrid screening and identified that the auxin transporter WALLS ARE THIN1 (WAT1) interacts with PagARGOS. Overexpression of PagARGOS up-regulated WAT1, activating a downstream auxin response promoting cambial cell division and xylem differentiation for wood formation. Moreover, overexpressing PagARGOS caused not only higher wood yield but also lower lignin content compared with wild-type controls. PagARGOS is therefore a potential candidate gene for engineering fast-growing and low-lignin trees with improved biomass production.

Epigenetic regulation and epigenetic memory resetting during plant rejuvenation.

Reversal of plant developmental status from the mature to the juvenile phase, thus leading to the restoration of the developmental potential, is referred to as plant rejuvenation. It involves multilayer regulation, including resetting gene expression patterns, chromatin remodeling, and histone modifications, eventually resulting in the restoration of juvenile characteristics. Although plants can be successfully rejuvenated using some forestry practices to restore juvenile morphology, physiology, and reproductive capabilities, studies on the epigenetic mechanisms underlying this process are in the nascent stage. This review provides an overview of the plant rejuvenation process and discusses the key epigenetic mechanisms involved in DNA methylation, histone modification, and chromatin remodeling in the process of rejuvenation, as well as the roles of small RNAs in this process. Additionally, we present new inquiries regarding the epigenetic regulation of plant rejuvenation, aiming to advance our understanding of rejuvenation in sexually and asexually propagated plants. Overall, we highlight the importance of epigenetic mechanisms in the regulation of plant rejuvenation, providing valuable insights into the complexity of this process.

DAL10 is a direct target of putative DAL1-mediated age pathway in conifers.

The optimal timing of the transition from vegetative growth to reproductive growth is critical for plant reproductive success, and the underlying regulatory mechanisms have been well studied in angiosperm model species, but relatively little in gymnosperms. DAL1, a MADS domain transcription factor (TF) gene that shows a conserved age-related expression profile in conifers, may be an age timer. However, how the DAL1 mediates the onset of reproductive growth remains poorly understood. Here, we have shown that the PtDAL1 directly regulates the PtDAL10 transcription by binding to its promoter region in vitro. PtDAL1, forms ternary complexes in vitro and in N. benthamiana with PtDAL10 and PtMADS11, two potential candidate regulators of the vegetative to reproductive transition in Chinese pine (Pinus tabuliformis). The PtDAL10 was progressively induced in new shoots with age and highly accumulated in male and female cones. Overexpression of PtDAL10 rescued the flowering of ft-10 and soc1-1-2 mutants in Arabidopsis. We provide insight into the molecular components associated with the PtDAL1, which integrates the vegetative to reproductive phase transition into age-mediated progressive development of the whole plant in conifers.

The JASMONATE ZIM-domain-OPEN STOMATA1 cascade integrates jasmonic acid and abscisic acid signaling to regulate drought tolerance by mediating stomatal closure in poplar.

Drought, which directly affects the yield of crops and trees, is a natural stress with a profound impact on the economy. Improving water use efficiency (WUE) and drought tolerance are relatively effective strategies to alleviate drought stress. OPEN STOMATA1 (OST1), at the core of abscisic acid (ABA) signaling, can improve WUE by regulating stomatal closure and photosynthesis. Methyl jasmonate (MeJA) and ABA crosstalk is considered to be involved in the response to drought stress, but the detailed molecular mechanism is insufficiently known. Here, Populus euphratica, which naturally grows in arid and semiarid regions, was selected as the species for studying MeJA and ABA crosstalk under drought. A yeast two-hybrid assay was performed using PeOST1 as bait and a nucleus-localized factor, JASMONATE ZIM-domain protein 2 (PeJAZ2), was found to participate in MeJA signaling by interacting with PeOST1. Overexpression of PeJAZ2 in poplar notably increased water deficit tolerance and WUE in both severe and mild drought stress by regulating ABA signaling rather than ABA synthesis. Furthermore, a PeJAZ2 overexpression line was shown to have greater ABA-induced stomatal closure and hydrogen peroxide (H2O2) production. Collectively, this evidence establishes a mechanism in which PeJAZ2 acts as a positive regulator in response to drought stress via ABA-induced stomatal closure caused by H2O2 production. Our study presents a new insight into the crosstalk of ABA and jasmonic acid signaling in regulating WUE and drought stress, providing a basis of the drought tolerance mechanism of P. euphratica.

Genome-wide TCP transcription factors analysis provides insight into their new functions in seasonal and diurnal growth rhythm in Pinus tabuliformis.

BACKGROUND: Pinus tabuliformis adapts to cold climate with dry winter in northern China, serving as important commercial tree species. The TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTOR family(TCP)transcription factors were found to play a role in the circadian clock system in Arabidopsis. However, the role of TCP transcription factors in P. tabuliformis remains little understood. RESULTS: In the present study, 43 TCP genes were identified from P. tabuliformis genome database. Based on the phylogeny tree and sequence similarity, the 43 TCP genes were classified into four groups. The motif results showed that different subfamilies indeed contained different motifs. Clade II genes contain motif 1, clade I genes contain motif 1, 8, 10 and clade III and IV contain more motifs, which is consistent with our grouping results. The structural analysis of PtTCP genes showed that most PtTCPs lacked introns. The distribution of clade I and clade II on the chromosome is relatively scattered, while clade III and clade IV is relatively concentrated. Co-expression network indicated that PtTCP2, PtTCP12, PtTCP36, PtTCP37, PtTCP38, PtTCP41 and PtTCP43 were co-expressed with clock genes in annual cycle and their annual cycle expression profiles both showed obvious seasonal oscillations. PtTCP2, PtTCP12, PtTCP37, PtTCP38, PtTCP40, PtTCP41, PtTCP42 and PtTCP43 were co-expressed with clock genes in diurnal cycle. Only the expression of PtTCP42 showed diurnal oscillation. CONCLUSIONS: The TCP gene family, especially clade II, may play an important role in the regulation of the season and circadian rhythm of P. tabuliformis. In addition, the low temperature in winter may affect the diurnal oscillations.

MADS-box transcription factors MADS11 and DAL1 interact to mediate the vegetative-to-reproductive transition in pine.

The reproductive transition is an important event that is crucial for plant survival and reproduction. Relative to the thorough understanding of the vegetative phase transition in angiosperms, a little is known about this process in perennial conifers. To gain insight into the molecular basis of the regulatory mechanism in conifers, we used temporal dynamic transcriptome analysis with samples from seven different ages of Pinus tabuliformis to identify a gene module substantially associated with aging. The results first demonstrated that the phase change in P. tabuliformis occurred as an unexpectedly rapid transition rather than a slow, gradual progression. The age-related gene module contains 33 transcription factors and was enriched in genes that belong to the MADS (MCMl, AGAMOUS, DEFICIENS, SRF)-box family, including six SOC1-like genes and DAL1 and DAL10. Expression analysis in P. tabuliformis and a late-cone-setting P. bungeana mutant showed a tight association between PtMADS11 and reproductive competence. We then confirmed that MADS11 and DAL1 coordinate the aging pathway through physical interaction. Overexpression of PtMADS11 and PtDAL1 partially rescued the flowering of 35S::miR156A and spl1,2,3,4,5,6 mutants in Arabidopsis (Arabidopsis thaliana), but only PtMADS11 could rescue the flowering of the ft-10 mutant, suggesting PtMADS11 and PtDAL1 play different roles in flowering regulatory networks in Arabidopsis. The PtMADS11 could not alter the flowering phenotype of soc1-1-2, indicating it may function differently from AtSOC1 in Arabidopsis. In this study, we identified the MADS11 gene in pine as a regulatory mediator of the juvenile-to-adult transition with functions differentiated from the angiosperm SOC1.

Gibberellin Signaling Is Required for Far-Red Light-Induced Shoot Elongation in Pinus tabuliformis Seedlings.

Gibberellin (GA) is known to play an important role in low red/far-red (R:FR) light ratio-mediated hypocotyl and petiole elongation in Arabidopsis (Arabidopsis thaliana). However, the regulatory relationship between low R:FR and GAs remains unclear, especially in gymnosperms. To increase our understanding of the molecular basis of low R:FR-mediated shoot elongation in pines and to determine whether there is an association between low R:FR and GAs action, we explored the morphological and transcriptomic changes triggered by low R:FR, GAs, and paclobutrazol (PAC), a GAs biosynthesis inhibitor, in Pinus tabuliformis seedlings. Transcriptome profiles revealed that low R:FR conditions and GAs have a common set of transcriptional targets in P. tabuliformis We provide evidence that the effect of low R:FR on shoot elongation in P. tabuliformis is at least partially modulated by GAs accumulation, which can be largely attenuated by PAC. GAs are also involved in the cross talk between different phytohormones in the low R:FR response. A GA biosynthesis gene, encoding ent-kaurenoic acid oxidase (KAO), was strongly stimulated by low R:FR without being affected by GAs feedback regulation or the photoperiod. We show that GA signaling is required for low R:FR-induced shoot elongation in P tabuliformis seedlings, and that there are different regulatory targets for low R:FR-mediated GA biosynthesis between conifers and angiosperms.

Transcriptome-wide isolation and expression of NF-Y gene family in male cone development and hormonal treatment of Pinus tabuliformis.

It is known that nuclear factor Y (NF-Y) transcription factors play an important role in flowering time regulation and hormone response (ABA, GA) in angiosperms, but, little known in conifers. Moreover, the NF-Y gene family has not been comprehensively reported in conifers. Here, we identified 9 NF-YA, 9 NF-YB and 10 NF-YC genes in Pinus tabuliformis using Arabidopsis NF-Y protein sequences as queries. Additionally, by comparing conserved regions and phylogenetic relationships of the PtNF-Ys, we found that NF-Ys were both conserved and altered during evolution. PtTFL2, PtCO, PtNF-YC1 and PtNF-YC4 were exploited by expression profile in male cone development and correlation analysis. Furthermore, NF-YC1/4 and DPL (DELLA protein of P. tabuliformis) were interacted by yeast two-hybrid and BiFC assays, which suggested that NF-YC1/4 may be involved in gibberellins signaling pathway. Moreover, the multiple types of phytohormones-responsive cis-elements (ABA, JA, IAA, SA) have been found, and gene expression profile analysis showed that many NF-Y genes responded positively to SA and as opposed to IAA and JA, revealing the potential role of NF-Ys in conifers resistance. In summary, this study provided the basis for further investigation of the function of NF-Y genes in conifers.

The Transcriptional Landscape and Hub Genes Associated with Physiological Responses to Drought Stress in Pinus tabuliformis.

Drought stress has an extensive impact on regulating various physiological, metabolic, and molecular responses. In the present study, the Pinus tabuliformis transcriptome was studied to evaluate the drought-responsive genes using RNA- Sequencing approache. The results depicted that photosynthetic rate and H2O conductance started to decline under drought but recovered 24 h after re-watering; however, the intercellular CO2 concentration (Ci) increased with the onset of drought. We identified 84 drought-responsive transcription factors, 62 protein kinases, 17 transcriptional regulators, and 10 network hub genes. Additionally, we observed the expression patterns of several important gene families, including 2192 genes positively expressed in all 48 samples, and 40 genes were commonly co-expressed in all drought and recovery stages compared with the control samples. The drought-responsive transcriptome was conserved mainly between P. tabuliformis and A. thaliana, as 70% (6163) genes had a homologous in arabidopsis, out of which 52% homologous (3178 genes corresponding to 2086 genes in Arabidopsis) were also drought response genes in arabidopsis. The collaborative network exhibited 10 core hub genes integrating with ABA-dependent and independent pathways closely conserved with the ABA signaling pathway in the transcription factors module. PtNCED3 from the ABA family genes had shown significantly different expression patterns under control, mild, prolonged drought, and recovery stages. We found the expression pattern was considerably increased with the prolonged drought condition. PtNCED3 highly expressed in all drought-tested samples; more interestingly, expression pattern was higher under mild and prolonged drought. PtNCED3 is reported as one of the important regulating enzymes in ABA synthesis. The continuous accumulation of ABA in leaves increased resistance against drought was due to accumulation of PtNCED3 under drought stress in the pine needles.

Comparative transcriptome analyses reveal two distinct transcriptional modules associated with pollen shedding time in pine.

BACKGROUND: Seasonal flowering time is an ecologically and economically important trait in temperate trees. Previous studies have shown that temperature in many tree species plays a pivotal role in regulating flowering time. However, genetic control of flowering time is not synchronised in different individual trees under comparable temperature conditions, the underlying molecular mechanism is mainly to be investigated. RESULTS: In the present study, we analysed the transcript abundance in male cones and needles from six early pollen-shedding trees (EPs) and six neighbouring late pollen-shedding trees (LPs) in Pinus tabuliformis at three consecutive time points in early spring. We found that the EPs and LPs had distinct preferred transcriptional modules in their male cones and, interestingly, the expression pattern was also consistently maintained in needles even during the winter dormancy period. Additionally, the preferred pattern in EPs was also adopted by other fast-growing tissues, such as elongating new shoots. Enhancement of nucleic acid synthesis and stress resistance pathways under cold conditions can facilitate rapid growth and maintain higher transcriptional activity. CONCLUSIONS: During the cold winter and early spring seasons, the EPs were more sensitive to relatively warmer temperatures and showed higher transcriptomic activity than the LPs, indicating that EPs required less heat accumulation for pollen shedding than LPs. These results provided a transcriptomic-wide understanding of the temporal regulation of pollen shedding in pines.

A transcriptomics investigation into pine reproductive organ development.

The development of reproductive structures in gymnosperms is still poorly studied because of a lack of genomic information and useful genetic tools. The hermaphroditic reproductive structure derived from unisexual gymnosperms is an even less studied aspect of seed plant evolution. To extend our understanding of the molecular mechanism of hermaphroditism and the determination of sexual identity of conifer reproductive structures in general, unisexual and bisexual cones from Pinus tabuliformis were profiled for gene expression using 60K microarrays. Expression patterns of genes during progression of sexual cone development were analysed using RNA-seq. The results showed that, overall, the transcriptomes of male structures in bisexual cones were more similar to those of female cones. However, the expression of several MADS-box genes in the bisexual cones was similar to that of male cones at the more juvenile developmental stage, while despite these expression shifts, male structures of bisexual cones and normal male cones were histologically indistinguishable and cone development was continuous. This study represents a starting point for in-depth analysis of the molecular regulation of cone development and also the origin of hermaphroditism in pine.

Identification and expression profiles of sRNAs and their biogenesis and action-related genes in male and female cones of Pinus tabuliformis.

BACKGROUND: Small RNA (sRNA) play pivotal roles in reproductive development, and their biogenesis and action mechanisms are well characterised in angiosperm plants; however, corresponding studies in conifers are very limited. To improve our understanding of the roles of sRNA pathways in the reproductive development of conifers, the genes associated with sRNA biogenesis and action pathways were identified and analysed, and sRNA sequencing and parallel analysis of RNA ends (PARE) were performed in male and female cones of the Chinese pine (Pinus tabuliformis). RESULTS: Based on high-quality reference transcriptomic sequences, 21 high-confidence homologues involved in sRNA biogenesis and action in P. tabuliformis were identified, including two different DCL3 genes and one AGO4 gene. More than 75 % of genes involved in sRNA biogenesis and action have higher expression levels in female than in male cones. Twenty-six microRNA (miRNA) families and 74 targets, including 46 24-nt sRNAs with a 5' A, which are specifically expressed in male cones or female cones and probably bind to AGO4, were identified. CONCLUSIONS: The sRNA pathways have higher activity in female than in male cones, and the miRNA pathways are the main sRNA pathways in P. tabuliformis. The low level of 24-nt short-interfering RNAs in conifers is not caused by the absence of biogenesis-related genes or AGO-binding proteins, but most likely caused by the low accumulation of these key components. The identification of sRNAs and their targets, as well as genes associated with sRNA biogenesis and action, will provide a good starting point for investigations into the roles of sRNA pathways in cone development in conifers.

Isolation and expression profiles of gibberellin metabolism genes in developing male and female cones of Pinus tabuliformis.

Gibberellins (GAs) are important in the floral regulatory networks of angiosperm plants. Several lines of evidence suggest that GAs also play a pivotal role in conifer male and female cone development. To gain new insights into the GA metabolism pathway in conifer trees and the role of GA metabolism in male and female cone development, we identified GA metabolism genes in Pinus tabuliformis. These included one PtCPS gene, one PtKS gene, one PtKO gene, TWO PtKAO genes, one PtGA20ox gene, two PtGA3ox genes and 12 PtGA2ox genes. According to phylogenetic analysis, the GA biosynthesis pathway evolved after the divergence of mosses from ferns, but the GA-deactivating gene family underwent divided expansion after divergence of the angiosperms from gymnosperms. However, the active sites of all GA metabolism enzymes were conserved during the evolution of land plants. During male and female cone development of P. tabuliformis, the expression of most of the PtGA2ox genes, especially PtGA2ox10, was higher than GA biosynthesis genes. However, the expression of PtKAO1 in cones peaked at a very early developmental stage. The expression pattern of GA metabolism genes indicated that GAs play different roles at the early and late stages of cone development.

Genome-wide identification of the Pinus tabuliformis CONSTANS-like gene family and their potential roles in reproductive cone development.

The CONSTANS-like (COL) genes, as a core transcription factor in the photoperiod regulation pathway, play a key role in plant reproduction development. However, their molecular characterization has rarely been studied in Pinus tabuliformis. Here, 10 PtCOL genes were identified in the P. tabuliformis genome and multiple sequence alignments have indicated that the PtCOL proteins contained highly conserved B-BOX1 and CCT domains. Sequence similarity analysis showed that PtCOL1 and PtCOL3 had the higher similarity with Norway spruce COLs (PaCOL2 and PaCOL1) and Arabidopsis COLs (AtCOL3, 4 and 5), respectively. Phylogeny and gene structure analyses revealed that PtCOLs were divided into three subgroups, each with identical or similar distributions of exons, introns, and motifs. Moreover, 10 PtCOLs were distributed on 6 chromosomes and PtCOL9 has syntenic gene pairs in both Ginkgo biloba and Sequoiadendron giganteum. Interestingly, in transcriptome profiles, most PtCOLs exhibited a diurnal oscillation pattern under both long (LD) and short (SD) day conditions. Additionally, PtCOLs were highly expressed in needles and female cones, and showed different spatial expression patterns. Among the ten PtCOLs, PtCOL1/3 heterologous overexpression Arabidopsis displayed a delayed-flowering phenotype under SD, indicating that they are likely to play a crucial role in the reproductive development. Additionally, PtCOL1 and PtCOL3 were not only capable of interacting with each other, but they were each capable of interacting with themselves. Furthermore, PtCOL1 and PtCOL3 were also involved in the MADS-box protein-protein interaction (PPI) network in P. tabuliformis cone development. Direct interactions of PtDAL11 with PtCOL1/3 impeded PtCOL1/3 translocation into the nucleus. In summary, this study provided comprehensive understanding for the functions of the PtCOL gene family and revealed their biological roles in the photoperiod-dependent P. tabuliformis cone development.

"Point by point" source: The Chinese pine plantations in North China by evidence from mtDNA.

The geographical variation and domestication of tree species are an important part of the theory of forest introduction, and the tracing of the germplasm is the theoretical basis for the establishment of high-quality plantations. Chinese pine (Pinus tabuliformis Carr.) is an important native timber tree species widely distributed in northern China, but it is unclear exactly where germplasm of the main Chinese pine plantation populations originated. Here, using two mtDNA markers, we analyzed 796 individuals representing 35 populations (matR marker), and 873 individuals representing 38 populations (nad5-1 marker) of the major natural and artificial populations in northern China, respectively (Shanxi, Hebei and Liaoning provinces). The results confirmed that the core position of natural SX* populations ("*" means natural population) in the Chinese pine populations of northern China, the genetic diversity of HB and LN plantations was higher than that of natural SX* populations, and there was a large difference in genetic background within the groups of SX* and LN, HB showed the opposite. More importantly, we completed the "point by point" tracing of the HB and LN plantings. The results indicated that almost all HB populations originated from SX* (GDS*, ZTS*, GCS*, and THS*), which resulted in homogeneity of the genetic background of HB populations. Most of germplasm of the LN plantations originated from LN* (ZJS* and WF*), and the other part originated from GDS* (SX*), resulting in the large differences in the genetic background within the LN group. Our results provided a reliable theoretical basis for the scientific allocation, management, and utilization of Chinese pine populations in northern China, and for promoting the high-quality establishment of Chinese pine plantations.

Transcriptome characterisation of Pinus tabuliformis and evolution of genes in the Pinus phylogeny.

BACKGROUND: The Chinese pine (Pinus tabuliformis) is an indigenous conifer species in northern China but is relatively underdeveloped as a genomic resource; thus, limiting gene discovery and breeding. Large-scale transcriptome data were obtained using a next-generation sequencing platform to compensate for the lack of P. tabuliformis genomic information. RESULTS: The increasing amount of transcriptome data on Pinus provides an excellent resource for multi-gene phylogenetic analysis and studies on how conserved genes and functions are maintained in the face of species divergence. The first P. tabuliformis transcriptome from a normalised cDNA library of multiple tissues and individuals was sequenced in a full 454 GS-FLX run, producing 911,302 sequencing reads. The high quality overlapping expressed sequence tags (ESTs) were assembled into 46,584 putative transcripts, and more than 700 SSRs and 92,000 SNPs/InDels were characterised. Comparative analysis of the transcriptome of six conifer species yielded 191 orthologues, from which we inferred a phylogenetic tree, evolutionary patterns and calculated rates of gene diversion. We also identified 938 fast evolving sequences that may be useful for identifying genes that perhaps evolved in response to positive selection and might be responsible for speciation in the Pinus lineage. CONCLUSIONS: A large collection of high-quality ESTs was obtained, de novo assembled and characterised, which represents a dramatic expansion of the current transcript catalogues of P. tabuliformis and which will gradually be applied in breeding programs of P. tabuliformis. Furthermore, these data will facilitate future studies of the comparative genomics of P. tabuliformis and other related species.

Proper gibberellin localization in vascular tissue is required to regulate adventitious root development in tobacco.

Bioactive gibberellins (GAs) are involved in many developmental aspects of the life cycle of plants, acting either directly or through interaction with other hormones. Accumulating evidence suggests that GAs have an important effect on root growth; however, there is currently little information on the specific regulatory mechanism of GAs during adventitious root development. A study was conducted on tobacco (Nicotiana tabacum) plants for altered rates of biosynthesis, catabolism, and GA signalling constitutively or in specific tissues using a transgenic approach. In the present study, PtGA20ox, PtGA2ox1, and PtGAI were overexpressed under the control of the 35S promoter, vascular cambium-specific promoter (LMX5), or root meristem-specific promoter (TobRB7), respectively. Evidence is provided that the precise localization of bioactive GA in the stem but not in the roots is required to regulate adventitious root development in tobacco. High levels of GA negatively regulate the early initiation step of root formation through interactions with auxin, while a proper and mobile GA signal is required for the emergence and subsequent long-term elongation of established primordia. The results demonstrated that GAs have an inhibitory effect on adventitious root formation but a stimulatory effect on root elongation.