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Multiple plant hormones, including strigolactone (SL), play key roles in regulating flowering time. The Arabidopsis (Arabidopsis thaliana) DWARF14 (AtD14) receptor perceives SL and recruits F-box protein MORE AXILLARY GROWTH2 (MAX2) and the SUPPRESSOR OF MAX2-LIKE (SMXL) family proteins. These interactions lead to the degradation of the SMXL repressor proteins, thereby regulating shoot branching, leaf shape, and other developmental processes. However, the molecular mechanism by which SL regulates plant flowering remains elusive. Here, we demonstrate that intact strigolactone biosynthesis and signaling pathways are essential for normal flowering in Arabidopsis. Loss-of-function mutants in both SL biosynthesis (max3) and signaling (Atd14 and max2) pathways display earlier flowering, whereas the repressor triple mutant smxl6/7/8 (s678) exhibits the opposite phenotype. Retention of AtD14 in the cytoplasm leads to its inability to repress flowering. Moreover, we show that nuclear-localized AtD14 employs dual strategies to enhance the function of the AP2 transcription factor TARGET OF EAT1 (TOE1). AtD14 directly binds to TOE1 in an SL-dependent manner and stabilizes it. In addition, AtD14-mediated degradation of SMXL7 releases TOE1 from the repressor protein, allowing it to bind to and inhibit the FLOWERING LOCUS T (FT) promoter. This results in reduced FT transcription and delayed flowering. In summary, AtD14 perception of SL enables the transcription factor TOE1 to repress flowering, providing insights into hormonal control of plant flowering., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Strigolactones (SLs), a class of carotenoid-derived hormones, play a crucial role in flowering plants by regulating underground communication with symbiotic arbuscular mycorrhizal fungi (AM) and controlling shoot and root architecture. While the functions of core SL genes have been characterized in many plants, their roles in non-tracheophyte plants like liverworts require further investigation. In this study, we employed the model liverwort species Marchantia polymorpha , which lacks detectable SL production and orthologs of key SL biosynthetic genes, including CAROTENOID CLEAVAGE DIOXYGENASE 8 ( CCD8 ) and MORE AXILLARY GROWTH 1 ( MAX1 ). However, it retains some SL pathway components, including DWARF27 ( D27 ) and CCD7 . To help elucidate the function of these remaining components in M. polymorpha , knockout mutants were generated for MpD27-1 , MpD27-2 and MpCCD7 . Phenotypic comparisons of these mutants with the wild-type control revealed a novel role for these genes in regulating the release of gemmae from the gemma cup and the germination and growth of gemmae in the dark. Mpd27-1 , Mpd27-2 , and Mpccd7 mutants showed lower transcript abundance of genes involved in photosynthesis, such as EARLY LIGHT INDUCED ( ELI ), and stress responses such as LATE EMBRYOGENESIS ABUNDANT ( LEA ) but exhibited higher transcript levels of ETHYLENE RESPONSE FACTORS ( ERFs ) and SL and carotenoid related genes, such as TERPENE SYNTHASE ( TS ), CCD7 and LECITHIN-RETINAL ACYL TRANSFERASE (LRAT) . Furthermore, the mutants of M. polymorpha in the SL pathway exhibited increased contents of carotenoid. This unveils a previously unrecognized role for MpD27-1, MpD27-2 and MpCCD7 in controlling release, germination, and growth of gemmae in response to varying light conditions. These discoveries enhance our comprehension of the regulatory functions of SL biosynthesis genes in non-flowering plants., Competing Interests: Authors RJ, JT, CA, BJ, RD, NA, YZ, KD and KS were employed by the company The New Zealand Institute for Plant and Food Research Limited. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Jibran, Tahir, Andre, Janssen, Drummond, Albert, Zhou, Davies and Snowden.)

The action of the petunia strigolactone (SL) hormone receptor DAD2 is dependent not only on its interaction with the PhMAX2A and PhD53A proteins, but also on its expression patterns within the plant. Previously, in a yeast-2-hybrid system, we showed that a series of a single and double amino acid mutants of DAD2 had altered interactions with these binding partners. In this study, we tested the mutants in two plant systems, Arabidopsis and petunia. Testing in Arabidopsis was enabled by creating a CRISPR-Cas9 knockout mutant of the Arabidopsis strigolactone receptor (AtD14). We produced SL receptor activity in both systems using wild type and mutant genes; however, the mutants had functions largely indistinguishable from those of the wild type. The expression of the wild type DAD2 from the CaMV 35S promoter in dad2 petunia produced plants neither quite like the dad2 mutant nor the V26 wild type. These plants had greater height and leaf size although branch number and the plant shape remained more like those of the mutant. These traits may be valuable in the context of a restricted area growing system such as controlled environment agriculture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Drummond, Lee, Luo, Dakin, Janssen and Snowden.)

Background: Shoot branching of flowering plants exhibits phenotypic plasticity and variability. This plasticity is determined by the activity of axillary meristems, which in turn is influenced by endogenous and exogenous cues such as nutrients and light. In many species, not all buds on the main shoot develop into branches despite favorable growing conditions. In petunia, basal axillary buds (buds 1-3) typically do not grow out to form branches, while more apical axillary buds (buds 6 and 7) are competent to grow., Results: The genetic regulation of buds was explored using transcriptome analyses of petunia axillary buds at different positions on the main stem. To suppress or promote bud outgrowth, we grew the plants in media with differing phosphate (P) levels. Using RNA-seq, we found many (> 5000) differentially expressed genes between bud 6 or 7, and bud 2. In addition, more genes were differentially expressed when we transferred the plants from low P to high P medium, compared with shifting from high P to low P medium. Buds 6 and 7 had increased transcript abundance of cytokinin and auxin-related genes, whereas the basal non-growing buds (bud 2 and to a lesser extent bud 3) had higher expression of strigolactone, abscisic acid, and dormancy-related genes, suggesting the outgrowth of these basal buds was actively suppressed. Consistent with this, the expression of ABA associated genes decreased significantly in apical buds after stimulating growth by switching the medium from low P to high P. Furthermore, comparisons between our data and transcriptome data from other species suggest that the suppression of outgrowth of bud 2 was correlated with a limited supply of carbon to these axillary buds. Candidate genes that might repress bud outgrowth were identified by co-expression analysis., Conclusions: Plants need to balance growth of axillary buds into branches to fit with available resources while allowing some buds to remain dormant to grow after the loss of plant parts or in response to a change in environmental conditions. Here we demonstrate that different buds on the same plant with different developmental potentials have quite different transcriptome profiles., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The architecture of flowering plants exhibits both phenotypic diversity and plasticity, determined, in part, by the number and activity of axillary meristems and, in part, by the growth characteristics of the branches that develop from the axillary buds. The plasticity of shoot branching results from a combination of various intrinsic and genetic elements, such as number and position of nodes and type of growth phase, as well as environmental signals such as nutrient availability, light characteristics, and temperature (Napoli et al., 1998; Bennett and Leyser, 2006; Janssen et al., 2014; Teichmann and Muhr, 2015; Ueda and Yanagisawa, 2019). Axillary meristem initiation and axillary bud outgrowth are controlled by a complex and interconnected regulatory network. Although many of the genes and hormones that modulate branching patterns have been discovered and characterized through genetic and biochemical studies, there are still many gaps in our understanding of the control mechanisms at play. In this review, we will summarize our current knowledge of the control of axillary meristem initiation and outgrowth into a branch., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Differential scanning fluorimetry (DSF) is a method used for assessing the interaction of ligands with proteins. In most cases binding of a ligand to proteins tends to increase the melting temperature (T m ) of the protein involved. However, in the case of strigolactone receptors (e.g., D14, AtD14, DAD2, RMS3) from plants, the T m tends to be reduced in the presence of strigolactones. This is likely due to increased flexibility of the receptors in the presence of hormone ligands.DSF experiments are simple, fast, amenable to high-throughput formats, and cost effective. They have therefore gained in popularity, including within the field of SL signaling. Typically in DSF the receptor protein is purified and incubated with the ligand (strigolactone, agonist, or antagonist) and a (fluorescent) reporter dye. The mixture is then placed in a quantitative PCR instrument and subjected to an increasing temperature gradient. Changes in fluorescence are recorded along the gradient, as the dye interacts with unfolded portions of the protein becoming accessible when the protein "melts". Differences in the temperature at which the protein unfolds in the absence and in the presence of the ligand are interpreted as indicating interactions between the ligand and the receptor.

Strigolactones (SLs) are terpenoid-derived plant hormones that regulate various developmental processes, particularly shoot branching, root development, and leaf senescence. The SL receptor has an unusual mode of action. Upon binding SL, it hydrolyzes the hormone, and then covalently binds one of the hydrolytic products. These initial events enable the SL receptor DAD2 (in petunia) to interact with the F-box protein PhMAX2A of the Skp-Cullin-F-box (SCF) complex and/or a repressor of SL signaling, PhD53A. However, it remains unclear how binding and hydrolysis structurally alters the SL receptor to enable its engagement with signaling partners. Here, we used mutagenesis to alter DAD2 and affect SL hydrolysis or DAD2's ability to interact with its signaling partners. We identified three DAD2 variants whose hydrolytic activity had been separated from the receptor's interactions with PhMAX2A or PhD53A. Two variants, DAD2 N242I and DAD2 F135A , having substitutions in the core α/β hydrolase-fold domain and the hairpin, exhibited hormone-independent interactions with PhMAX2A and PhD53A, respectively. Conversely, the DAD2 D166A variant could not interact with PhMAX2A in the presence of SL, but its interaction with PhD53A remained unaffected. Structural analyses of DAD2 N242I and DAD2 D166A revealed only small differences compared with the structure of the WT receptor. Results of molecular dynamics simulations of the DAD2 N242I structure suggested that increased flexibility is a likely cause for its SL-independent interaction with PhMAX2A. Our results suggest that PhMAX2A and PhD53A have distinct binding sites on the SL receptor and that its flexibility is a major determinant of its interactions with these two downstream regulators., (© 2020 Lee et al.)

Strigolactones (SLs) are multifunctional plant hormones regulating essential physiological processes affecting growth and development. In vascular plants, SLs are recognized by α/β hydrolase-fold proteins from the D14/DAD2 (Dwarf14/Decreased Apical Dominance 2) family in the initial step of the signaling pathway. We have previously discovered that N -phenylanthranilic acid derivatives (e.g. tolfenamic acid) are potent antagonists of SL receptors, prompting us to design quinazolinone and quinazolinedione derivatives (QADs and QADDs, respectively) as second-generation antagonists. Initial in silico docking studies suggested that these compounds would bind to DAD2, the petunia SL receptor, with higher affinity than the first-generation compounds. However, only one of the QADs/QADDs tested in in vitro assays acted as a competitive antagonist of SL receptors, with reduced affinity and potency compared with its N -phenylanthranilic acid 'parent'. X-ray crystal structure analysis revealed that the binding mode of the active QADD inside DAD2's cavity was not that predicted in silico , highlighting a novel inhibition mechanism for SL receptors. Despite a ∼10-fold difference in potency in vitro , the QADD and tolfenamic acid had comparable activity in planta , suggesting that the QADD compensates for lower potency with increased bioavailability. Altogether, our results establish this QADD as a novel lead compound towards the development of potent and bioavailable antagonists of SL receptors., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

An enantioselective synthesis of the phenyl ring-containing strioglactone, (-)-solanocol, is described. Application of a Dynamic Kinetic Resolution (DKR) in the stereo-defining step enabled a step-economical synthesis to be achieved, and allowed access to natural and non-natural enantiomers with equal facility. Results of seed germination assays and Differential Scanning Fluorimetry (DSF) measurements with the known strigolactone receptor protein, Decreased Apical Dominance 2 (DAD2), are reported.

The strigolactone (SL) family of plant hormones regulates a broad range of physiological processes affecting plant growth and development and also plays essential roles in controlling interactions with parasitic weeds and symbiotic fungi. Recent progress elucidating details of SL biosynthesis, signaling, and transport offers many opportunities for discovering new plant-growth regulators via chemical interference. Here, using high-throughput screening and downstream biochemical assays, we identified N -phenylanthranilic acid derivatives as potent inhibitors of the SL receptors from petunia (DAD2), rice (OsD14), and Arabidopsis (AtD14). Crystal structures of DAD2 and OsD14 in complex with inhibitors further provided detailed insights into the inhibition mechanism, and in silico modeling of 19 other plant strigolactone receptors suggested that these compounds are active across a large range of plant species. Altogether, these results provide chemical tools for investigating SL signaling and further define a framework for structure-based approaches to design and validate optimized inhibitors of SL receptors for specific plant targets., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Branching has a major influence on the overall shape and productivity of a plant. Strigolactones (SLs) have been identified as plant hormones that have a key role in suppressing the outgrowth of axillary meristems. CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes are integral to the biosynthesis of SLs and are well characterized in annual plants, but their role in woody perennials is relatively unknown. We identified CCD7 and CCD8 orthologues from apple and demonstrated that MdCCD7 and MdCCD8 are able to complement the Arabidopsis branching mutants max3 and max4 respectively, indicating conserved function. RNAi lines of MdCCD7 show reduced gene expression and increased branching in apple. We performed reciprocal grafting experiments with combinations of MdCCD7 RNAi and wild-type 'Royal Gala' as rootstocks and scion. Unexpectedly, wild-type roots were unable to suppress branching in MdCCD7 RNAi scions. Another key finding was that MdCCD7 RNAi scions initiated phytomers at an increased rate relative to the wild type, resulting in a greater node number and primary shoot length. We suggest that localized SL biosynthesis in the shoot, rather than roots, controls axillary bud outgrowth and shoot growth rate in apple.

Background: Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models., Results: A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models., Conclusions: Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.

Plants alter their development in response to changes in their environment. This responsiveness has proven to be a successful evolutionary trait. Here, we tested the hypothesis that two key environmental factors, light and nutrition, are integrated within the axillary bud to promote or suppress the growth of the bud into a branch. Using petunia (Petunia hybrida) as a model for vegetative branching, we manipulated both light quality (as crowding and the red-to-far-red light ratio) and phosphate availability, such that the axillary bud at node 7 varied from deeply dormant to rapidly growing. In conjunction with the phenotypic characterization, we also monitored the state of the strigolactone (SL) pathway by quantifying SL-related gene transcripts. Mutants in the SL pathway inhibit but do not abolish the branching response to these environmental signals, and neither signal is dominant over the other, suggesting that the regulation of branching in response to the environment is complex. We have isolated three new putatively SL-related TCP (for Teosinte branched1, Cycloidia, and Proliferating cell factor) genes from petunia, and have identified that these TCP-type transcription factors may have roles in the SL signaling pathway both before and after the reception of the SL signal at the bud. We show that the abundance of the receptor transcript is regulated by light quality, such that axillary buds growing in added far-red light have greatly increased receptor transcript abundance. This suggests a mechanism whereby the impact of any SL signal reaching an axillary bud is modulated by the responsiveness of these cells to the signal., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Axillary meristems are formed in leaf axils and their growth into branches is a highly controlled process that is an important contributor to plant architecture. Here we discuss work that improves our understanding of the initiation and growth of axillary meristems. Recent results have implicated brassinosteroid signalling in the formation of axillary meristems. Our knowledge of axillary meristem outgrowth has also advanced, particularly in the areas of strigolactone signal production and perception, which have been shown to respond to environmental inputs. Auxins and cytokinins have also been linked to the control of axillary shoot development, revealing a complex network of signals that combine to regulate the outgrowth of an axillary meristem into a branch., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The signaling molecules strigolactone (SL) and karrikin are involved in seed germination, development of axillary meristems, senescence of leaves, and interactions with arbuscular mycorrhizal fungi. The signal transduction pathways for both SLs and karrikins require the same F-box protein (MAX2) and closely related α/β hydrolase fold proteins (DAD2 and KAI2). The crystal structure of DAD2 has been solved revealing an α/β hydrolase fold protein with an internal cavity capable of accommodating SLs. DAD2 responds to the SL analog GR24 by changing conformation and binding to MAX2 in a GR24 concentration-dependent manner. DAD2 can also catalyze hydrolysis of GR24. Structure activity relationships of analogs indicate that the butenolide ring common to both SLs and karrikins is essential for biological activity, but the remainder of the molecules can be significantly modified without loss of activity. The combination of data from the study of DAD2, KAI2, and chemical analogs of SLs and karrikins suggests a model for binding that requires nucleophilic attack by the active site serine of the hydrolase at the carbonyl atom of the butenolide ring. A conformational change occurs in the hydrolase that results in interaction with the F-box protein MAX2. Downstream signal transduction is then likely to occur via SCF (Skp-Cullin-F-box) complex-mediated ubiquitination of target proteins and their subsequent degradation. The role of the catalytic activity of the hydrolase is unclear but it may be integral in binding as well as possibly allowing the signal to be cleared from the receptor. The α/β hydrolase fold family consists mostly of active enzymes, with a few notable exceptions. We suggest that DAD2 and KAI2 represent an intermediate stage where some catalytic activity is retained at the same time as a receptor role has evolved.

Strigolactones are a recently discovered class of plant hormone involved in branching, leaf senescence, root development, and plant-microbe interactions. They are carotenoid-derived lactones, synthesized in the roots and transported acropetally to modulate axillary bud outgrowth (i.e., branching). However, a receptor for strigolactones has not been identified. We have identified the DAD2 gene from petunia, an ortholog of the rice and Arabidopsis D14 genes, and present evidence for its roles in strigolactone perception and signaling. DAD2 acts in the strigolactone pathway, and the dad2 mutant is insensitive to the strigolactone analog GR24. The crystal structure of DAD2 reveals an α/β hydrolase fold containing a canonical catalytic triad with a large internal cavity capable of accommodating strigolactones. In the presence of GR24 DAD2 interacts with PhMAX2A, a central component of strigolactone signaling, in a GR24 concentration-dependent manner. DAD2 can hydrolyze GR24, with mutants of the catalytic triad abolishing both this activity and the ability of DAD2 to interact with PhMAX2A. The hydrolysis products can neither stimulate the protein-protein interaction nor modulate branching. These observations suggest that DAD2 acts to bind the mobile strigolactone signal and then interacts with PhMAX2A during catalysis to initiate an SCF-mediated signal transduction pathway., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Analysis of mutants with increased branching has revealed the strigolactone synthesis/perception pathway which regulates branching in plants. However, whether variation in this well conserved developmental signaling system contributes to the unique plant architectures of different species is yet to be determined. We examined petunia orthologs of the ArabidopsisMAX1 and MAX2 genes to characterize their role in petunia architecture. A single ortholog of MAX1, PhMAX1 which encodes a cytochrome P450, was identified and was able to complement the max1 mutant of Arabidopsis. Petunia has two copies of the MAX2 gene, PhMAX2A and PhMAX2B which encode F-Box proteins. Differences in the transcript levels of these two MAX2-like genes suggest diverging functions. Unlike PhMAX2B, PhMAX2A mRNA levels change in leaves of differing age/position on the plant. Nonetheless, this gene functionally complements the Arabidopsismax2 mutant indicating that the biochemical activity of the PhMAX2A protein is not significantly different from MAX2. The expression of the petunia strigolactone pathway genes (PhCCD7, PhCCD8, PhMAX1, PhMAX2A, and PhMAX2B) was then further investigated throughout the development of wild-type petunia plants. Three of these genes showed changes in mRNA levels over a development series. Alterations to the expression patterns of these genes may influence the branching growth habit of plants by changing strigolactone production and/or sensitivity. These changes could allow both subtle and dramatic changes to branching within and between species.

• CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes have been demonstrated to play an integral role in the control of branch development in model plants, including Arabidopsis, pea (Pisum sativum), petunia (Petunia hybrida) and rice (Oryza sativa). • Actinidia chinensis is a woody perennial plant grown for commercial production of kiwifruit. CCD7 and CCD8 genes were isolated from A. chinensis and these genes are predominantly expressed in the roots of kiwifruit. AcCCD7 and AcCCD8 were able to complement the corresponding Arabidopsis mutants max3 and max4. The function of AcCCD8 in branch development was determined in transgenic kiwifruit plants containing an RNAi construct for AcCCD8. • Reduction in expression of AcCCD8 correlated with an increase in branch development and delayed leaf senescence. • The CCD pathway for control of branch development is conserved across a wide range of species, including kiwifruit, a woody perennial., (© The Authors (2010). Journal compilation © New Phytologist Trust (2010).)

Plants regulate the development of branches in response to environmental and developmental signals in order to maximize reproductive success. A number of hormone signals are involved in the regulation of branching and both their production and transmission affect axillary meristem outgrowth. With the identification of strigolactones as root-derived branch inhibitors it seems likely that a biochemical pathway starting from a carotenoid and resulting in production of a strigolactone hormone is present in most plants. Our observation that loss of CCD7 or CCD8 also results in production of a promoter of branching from roots shows the branching pathway has multiple levels of control which allows a high degree of sensitivity to subtle differences in environmental and developmental signals.

One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, are required for the production of a branch-suppressing plant hormone. Here, we report that the decreased apical dominance3 (dad3) mutant of petunia (Petunia hybrida) results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant, and grafted petunia suggests that in petunia, CCD7 and CCD8 are coordinately regulated. In contrast to observations in Arabidopsis (Arabidopsis thaliana), ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots, branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching.

Background: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs)., Results: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified., Conclusion: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Background: Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45-55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple., Results: Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes., Conclusion: Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development.

Control of branch development is a major determinant of architecture in plants. Branching in petunia (Petunia hybrida) is controlled by the DECREASED APICAL DOMINANCE (DAD) genes. Gene functions were investigated by plant grafting, morphology studies, double-mutant characterization, and gene expression analysis. Both dad1-1 and dad3 increased branching mutants can be reverted to a near-wild-type phenotype by grafting to a wild-type or a dad2 mutant root stock, indicating that both genes affect the production of a graft-transmissible substance that controls branching. Expression of the DAD1 gene in the stems of grafted plants, detected by quantitative reverse transcription-polymerase chain reaction correlates with the branching phenotype of the plants. The dad2-1 mutant cannot be reverted by grafting, indicating that this gene acts predominantly in the shoot of the plant. Double-mutant analysis indicates that the DAD2 gene acts in the same pathway as the DAD1 and DAD3 genes because the dad1-1dad2-1 and dad2-1dad3 double mutants are indistinguishable from the dad2-1 mutant. However, the dad1-1dad3 double mutant has an additive phenotype, with decreased height of the plants, delayed flowering, and reduced germination rates compared to the single mutants. This result, together with the observation that the dad1-1 and dad3 mutants cannot be reverted by grafting to each other, suggests that the DAD1 and DAD3 genes act in the same pathway, but not in a simple stepwise fashion.

The domestic apple (Malus domestica; also known as Malus pumila Mill.) has become a model fruit crop in which to study commercial traits such as disease and pest resistance, grafting, and flavor and health compound biosynthesis. To speed the discovery of genes involved in these traits, develop markers to map genes, and breed new cultivars, we have produced a substantial expressed sequence tag collection from various tissues of apple, focusing on fruit tissues of the cultivar Royal Gala. Over 150,000 expressed sequence tags have been collected from 43 different cDNA libraries representing 34 different tissues and treatments. Clustering of these sequences results in a set of 42,938 nonredundant sequences comprising 17,460 tentative contigs and 25,478 singletons, together representing what we predict are approximately one-half the expressed genes from apple. Many potential molecular markers are abundant in the apple transcripts. Dinucleotide repeats are found in 4,018 nonredundant sequences, mainly in the 5'-untranslated region of the gene, with a bias toward one repeat type (containing AG, 88%) and against another (repeats containing CG, 0.1%). Trinucleotide repeats are most common in the predicted coding regions and do not show a similar degree of sequence bias in their representation. Bi-allelic single-nucleotide polymorphisms are highly abundant with one found, on average, every 706 bp of transcribed DNA. Predictions of the numbers of representatives from protein families indicate the presence of many genes involved in disease resistance and the biosynthesis of flavor and health-associated compounds. Comparisons of some of these gene families with Arabidopsis (Arabidopsis thaliana) suggest instances where there have been duplications in the lineages leading to apple of biosynthetic and regulatory genes that are expressed in fruit. This resource paves the way for a concerted functional genomics effort in this important temperate fruit crop.

In the past decade, enormous progress has been made in our understanding of the molecular and genetic control of meristem growth, maintenance and differentiation into plant organs. Several model plants have contributed to our current knowledge of meristem function. Research using Petunia has had a substantial share in this progress. Integration of information obtained from this species gives clues about the common and diverged pathways underlying the formation and functioning of plant meristems.

Carotenoids and carotenoid cleavage products play an important and integral role in plant development. The Decreased apical dominance1 (Dad1)/PhCCD8 gene of petunia (Petunia hybrida) encodes a hypothetical carotenoid cleavage dioxygenase (CCD) and ortholog of the MORE AXILLARY GROWTH4 (MAX4)/AtCCD8 gene. The dad1-1 mutant allele was inactivated by insertion of an unusual transposon (Dad-one transposon), and the dad1-3 allele is a revertant allele of dad1-1. Consistent with its role in producing a graft-transmissible compound that can alter branching, the Dad1/PhCCD8 gene is expressed in root and shoot tissue. This expression is upregulated in the stems of the dad1-1, dad2, and dad3 increased branching mutants, indicating feedback regulation of the gene in this tissue. However, this feedback regulation does not affect the root expression of Dad1/PhCCD8. Overexpression of Dad1/PhCCD8 in the dad1-1 mutant complemented the mutant phenotype, and RNA interference in the wild type resulted in an increased branching phenotype. Other differences in phenotype associated with the loss of Dad1/PhCCD8 function included altered timing of axillary meristem development, delayed leaf senescence, smaller flowers, reduced internode length, and reduced root growth. These data indicate that the substrate(s) and/or product(s) of the Dad1/PhCCD8 enzyme are mobile signal molecules with diverse roles in plant development.

The monopodial shoot axis of petunia (Petunia hybrida Vilm) has two different patterns of branch development. Basal lateral branching develops acropetally and is limited to a discrete number of nodes that correlate with the late rosette phase of growth (Zone II). Two zones of suppressed buds immediately precede and follow this zone of branching. Apical branching occurs in response to flowering, develops in a basipetal direction, and is restricted to the distal-most nodes on the monopodial axis. When grown under a short-day regime, an extension to the basal branching zone occurs, and growth of the main shoot axis is retarded. The sym1 mutant has an overall decrease in basal lateral branching compared with wild type whereas the three dad mutants have increased basal branching. The dad1-1 and dad2-1 mutants have no initial zone of suppressed branching whereas the dad3 mutant has a similar Zone II to wild type, but with a greater potential to form branches within this zone. The dad1-1 mutant exhibits delayed flowering, but the dad1-1 sym1 double mutant flowers at a similar node number to wild-type and branching is similar to dad1-1 indicating that these two aspects of the mutant dad1-1 phenotype are independent.

Polar auxin transport is crucial for the regulation of auxin action and required for some light-regulated responses during plant development. We have found that two mutants of Arabidopsis-doc1, which displays altered expression of light-regulated genes, and tir3, known for its reduced auxin transport-have similar defects and define mutations in a single gene that we have renamed BIG. BIG is very similar to the Drosophila gene Calossin/Pushover, a member of a gene family also present in Caenorhabditis elegans and human genomes. The protein encoded by BIG is extraordinary in size, 560 kD, and contains several putative Zn-finger domains. Expression-profiling experiments indicate that altered expression of multiple light-regulated genes in doc1 mutants can be suppressed by elevated levels of auxin caused by overexpression of an auxin biosynthetic gene, suggesting that normal auxin distribution is required to maintain low-level expression of these genes in the dark. Double mutants of tir3 with the auxin mutants pin1, pid, and axr1 display severe defects in auxin-dependent growth of the inflorescence. Chemical inhibitors of auxin transport change the intracellular localization of the auxin efflux carrier PIN1 in doc1/tir3 mutants, supporting the idea that BIG is required for normal auxin efflux.

A large amount of diversity of architectural form is found among flowering plants, and an important aspect of this diversity is the wide variation, ranging from simple to complex, found among branching patterns in plant shoot systems. Historically, the control of bud outgrowth has been attributed to the presence of a growing shoot apex. The term "apical dominance" is used to indicate that the shoot tip exerts an inhibitory control over proximal axillary buds. Through decapitation and/or hormone manipulation experiments, this inhibition has been attributed to the phytohormones auxin and cytokinin. Recent studies with mutants demonstrating increased branching indicate important additional roles for organs apart from those in the shoot tip and for signals other than cytokinin and auxin. This chapter provides a critical review of branching with an emphasis toward bud outgrowth in a developmental context. This review provides a detailed synopsis of physiological, genetic, and molecular studies and approaches for the investigation of branching regulation in plants.

The identification of a spontaneous mutable Hf1 allele in Petunia hybrida provided an opportunity to isolate and characterize a novel transposable element. This 9.9 kb element has features in common with members of the Spm family, such as homologous terminal inverted repeats and a 3 bp target site direct duplication within the Hf1 gene. The element is named Petunia Spm-like (Psl). The footprints left by excising elements have been isolated from several germinal revertants and sequence analysis shows similarities to those left by other Spm family members. Southern analysis shows that the transposon is present at low copy number in the genome of different inbred lines and species of Petunia. The germinal excision frequency of Psl was 21-33% in outcross populations. The element appears to be very mobile somatically in the inbred line V26, with 38% of plants from an inbred population showing new Psl-hybridizing bands by Southern analysis. The high somatic and germinal excision frequency demonstrated by Psl suggests that this element may have utility for gene tagging in petunia.

A series of promoter-GUS fusion constructs containing a portion of the rice triosephosphate isomerase (tpi) promoter, the first tpi intron, and the gene encoding bacterial beta-glucuronidase (GUS) were made. These constructs were electroporated into rice protoplasts and transient expression was monitored. Inclusion of the first intron from the rice tpi gene enhanced expression of the GUS gene from the tpi promoter when it was placed 5' of the GUS gene. When the tpi intron was placed in the 3'-untranslated region no enhancement of GUS gene expression was observed, indicating the importance of position in intron-mediated enhancement of gene expression.

We have investigated the response of four Al-induced genes (wali1, -3, -4, and -5) from wheat (Triticum aestivum L.) to other stresses. The relative transcript levels of wali1 (encoding a plant metallothionein-like protein), wali3 and wali5 (putative Bowman-Birk proteinase inhibitors), and wali4 (phenylalanine ammonialyase) increased in root tips of wheat after 2-d treatments with toxic levels of all other metals tested (Cd, Fe, Zn, Cu, Ga, In, and La). The expression levels of wali1, -3, -4, and -5 also increased in the root tips of plants grown in the presence of low levels of Ca (10[mu]M). The transcript levels of wali1, -3, and -5 increased in wounded leaf and root tissue, whereas the transcript levels of wali4 increased only in wounded leaves. The site of expression of wali1, -3, and -5 in root tips was identified using in situ hybridization. wali1 was expressed predominantly in the meristematic tissue of the root tip, whereas wali3 and wali5 were expressed predominantly in the cortical tissue of the root. Some changes in the site of expression of these genes were evident in the roots of Al-treated plants.

Five different cDNAs (termed wali1 to wali5 for Wheat Aluminum Induced) whose expression was induced by Al stress have been isolated from the root tips of Al-treated wheat (Triticum aestivum L.) plants. Four of these genes were induced 24 to 96 h after Al treatment, and their expression is reduced when the Al is removed. Each of these four genes was induced by inhibitory levels of Al in two wheat cultivars--Warigal, an Al-sensitive cultivar, and Waalt, an Al-tolerant cultivar. The fifth gene (wali2) showed a complex bimodal pattern of induction and was induced by Al only in the sensitive cultivar. Comparison of the nucleotide sequences of these clones to those in the sequence data bases showed that wali4 is homologous to phenylalanine ammonia-lyase and wali1 is homologous to a group of plant proteins that are cysteine-rich and have homology to metallothioneins. wali2 encodes a novel protein with a repeating motif of cysteine amino acids. The remaining two wali clones (wali3 and wali5) encode related, cysteine-rich proteins that show no significant homology to any known sequences.