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The environment is seldom optimal for plant growth and changes in abiotic and biotic signals, including temperature, water availability, radiation and pests, induce plant responses to optimise survival. The New Zealand native plant species and close relative to Arabidopsis thaliana, Pachycladon cheesemanii grows under environmental conditions that are unsustainable for many plant species. Here we compare the responses of both plant species to different stressors (low temperature, salt and UV-B radiation) to help understand how P. cheesemanii can grow in such harsh environments. The stress transcriptomes were then determined and comparative transcriptome and network analyses discovered similar and unique responses within species between different stresses, and between the two plant species. A number of widely studied plant stress processes were highly conserved in A. thaliana and P. cheesemanii. However, in response to cold stress, Gene Ontology terms related to glycosinolate metabolism were only enriched in P. cheesemanii. Salt stress was associated with alteration of the cuticle and proline biosynthesis in A. thaliana and P. cheesemanii, respectively. Anthocyanin production may be a strategy to cope with UV-B radiation stress in P. cheesemanii only. These results allowed us to construct broad stress response pathways in A. thaliana and P. cheesemanii and identify possible novel plant strategies that help mitigate environmental stress.

Flowering of the reference legume Medicago truncatula is promoted by winter cold (vernalization) followed by long-day photoperiods (VLD) similar to winter annual Arabidopsis. However, Medicago lacks FLC and CO, key regulators of Arabidopsis VLD flowering. Most plants have two INHIBITOR OF GROWTH (ING) genes (ING1 and ING2), encoding proteins with an ING domain with two anti-parallel alpha-helices and a plant homeodomain (PHD) finger, but their genetic role has not been previously described. In Medicago, Mting1 gene-edited mutants developed and flowered normally, but an Mting2-1 Tnt1 insertion mutant and gene-edited Mting2 mutants had developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes, and smaller seeds and barrels. Mting2 mutants had reduced expression of activators of flowering, including the FT-like gene MtFTa1, and increased expression of the candidate repressor MtTFL1c, consistent with the delayed flowering of the mutant. MtING2 overexpression complemented Mting2-1, but did not accelerate flowering in wild type. The MtING2 PHD finger bound H3K4me2/3 peptides weakly in vitro, but analysis of gene-edited mutants indicated that it was dispensable to MtING2 function in wild-type plants. RNA sequencing experiments indicated that7000 genes are mis-expressed in the Mting2-1 mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP-seq analysis identified5000 novel H3K4me3 locations in the genome of Mting2-1 mutants compared to wild type R108. Overall, our mutant study has uncovered an important physiological role of a plant ING2 gene in development, flowering, and gene expression, which likely involves an epigenetic mechanism.

Phosphatidylethanolamine-binding protein (PEBP) genes regulate flowering and architecture in many plant species. Here, we study kiwifruit (Actinidia chinensis, Ac) PEBP genes with homology to BROTHER OF FT AND TFL1 (BFT). CRISPR-Cas9 was used to target AcBFT genes in wild-type and fast-flowering kiwifruit backgrounds. The editing construct was designed to preferentially target AcBFT2, whose expression is elevated in dormant buds. Acbft lines displayed an evergrowing phenotype and increased branching, while control plants established winter dormancy. The evergrowing phenotype, encompassing delayed budset and advanced budbreak after defoliation, was identified in multiple independent lines with edits in both alleles of AcBFT2. RNA-seq analyses conducted using buds from gene-edited and control lines indicated that Acbft evergrowing plants had a transcriptome similar to that of actively growing wild-type plants, rather than dormant controls. Mutations in both alleles of AcBFT2 did not promote flowering in wild-type or affect flowering time, morphology and fertility in fast-flowering transgenic kiwifruit. In summary, editing of AcBFT2 has the potential to reduce plant dormancy with no adverse effect on flowering, giving rise to cultivars better suited for a changing climate.

Actinidia arguta and related species produce small edible kiwifruit (kiwiberry) with attractive consumer features and high nutritional value. As a recently developed crop, kiwiberry can be improved further by classical breeding or genetic manipulation. The currently available Agrobacterium-mediated transformation protocol is only applicable to a limited number of kiwiberry genotypes such as the female cultivar ‘Hortgem Tahi’. We developed protocols for regeneration and Agrobacterium-mediated transformation of two A. arguta genotypes, female AA06-01 and male AA05-06, also applicable to ‘Hortgem Tahi’. Altered composition of basal salts in the callus-induction media was sufficient to prevent callus browning in AA06-01 and ‘Hortgem Tahi’, but not in AA05-06. Altering the hormone composition to avoid a prolonged callus stage was successfully used to prevent browning and induce shoot development in all three genotypes. Using our improved protocols, Agrobacterium-mediated transformation of a binary vector carrying the neomycin phosphotransferase II (nptII) expression cassette gave rise to kanamycin-resistant plants of both AA06-01 and AA05-06 and the presence of the nptII transgene was confirmed by genomic PCR. The improved protocols were also used to ectopically overexpress the anthocyanin-related transcription factor AcMYB110 in ‘Hortgem Tahi’ giving rise to purple callus with elevated anthocyanin accumulation. AcMYB110 expression and increased levels of anthocyanins were detected in mature leaves of established transgenic plants compared to controls, clearly demonstrating the important role for MYB110 in regulation of anthocyanin accumulation in kiwiberry. The protocols developed during this study provide tools for further functional analyses and genetic manipulation of kiwiberry genotypes. Using a newly developed transformation method we were able to generate transgenic lines of three Actinidia arguta genotypes and demonstrate an important role for MYB110 in anthocyanin accumulation in kiwiberry.

Environmentally-responsive genes can affect fruit red colour via the activation of MYB transcription factors. The apple B-box (BBX) gene, BBX33/CONSTANS-like 11 (COL11) has been reported to influence apple red-skin colour in a light- and temperature-dependent manner. To further understand the role of apple BBX genes, other members of the BBX family were examined for effects on colour regulation. Expression of 23 BBX genes in apple skin was analysed during fruit development. We investigated the diurnal rhythm of expression of the BBX genes, the anthocyanin biosynthetic genes and a MYB activator, MYB10. Transactivation assays on the MYB10 promoter, showed that BBX proteins 1, 17, 15, 35, 51, and 54 were able to directly function as activators. Using truncated versions of the MYB10 promoter, a key region was identified for activation by BBX1. BBX1 enhanced the activation of MYB10 and MdbHLH3 on the promoter of the anthocyanin biosynthetic gene DFR. In transformed apple lines, over-expression of BBX1 reduced internal ethylene content and altered both cyanidin concentration and associated gene expression. We propose that, along with environmental signals, the control of MYB10 expression by BBXs in ‘Royal Gala’ fruit involves the integration of the expression of multiple BBXs to regulate fruit colour.

Flowering time influences the yield and productivity of legume crops. Medicago truncatula is a reference temperate legume that, like the winter annual Arabidopsis thaliana, shows accelerated flowering in response to vernalization (extended cold) and long-day (LD) photoperiods (VLD). However, unlike A. thaliana, M. truncatula appears to lack functional homologs of core flowering time regulators CONSTANS (CO) and FLOWERING LOCUS C (FLC) which act upstream of the mobile florigen FLOWERING LOCUS T (FT). Medicago truncatula has three LD-induced FT-like genes (MtFTa1, MtFTb1, and MtFTb2) with MtFTa1 promoting M. truncatula flowering in response to VLD. Another photoperiodic regulator in A. thaliana, FE, acts to induce FT expression. It also regulates the FT transport pathway and is required for phloem development. Our study identifies a M. truncatula FE homolog Medtr6g444980 (MtFE) which complements the late flowering fe-1 mutant when expressed from the phloem-specific SUCROSE-PROTON SYMPORTER 2 (SUC2) promoter. Analysis of two M. truncatula Tnt1 insertional mutants indicate that MtFE promotes flowering in LD and VLD and growth in all conditions tested. Expression of MtFTa1, MtFTb1, and MtFTb2 are reduced in Mtfe mutant (NF5076), correlating with its delayed flowering. The NF5076 mutant plants are much smaller than wild type indicating that MtFE is important for normal plant growth. The second mutant (NF18291) displays seedling lethality, like strong fe mutants. We searched for mutants in MtFTb1 and MtFTb2 identifying a Mtftb2 knock out Tnt1 mutant (NF20803). However, it did not flower significantly later than wild type. Previously, yeast-two-hybrid assays (Y2H) suggested that Arabidopsis FE interacted with CO and NUCLEAR FACTOR-Y (NF-Y)-like proteins to regulate FT. We found that MtFE interacts with CO and also M. truncatula NF-Y-like proteins in Y2H experiments. Our study indicates that despite the apparent absence of a functional MtCO-like gene, M. truncatula FE likely influences photoperiodic FT expression and flowering time in M. truncatula via a partially conserved mechanism with A. thaliana.

Background Pachycladon cheesemanii is a close relative of Arabidopsis thaliana and is an allotetraploid perennial herb which is widespread in the South Island of New Zealand. It grows at altitudes of up to 1000 m where it is subject to relatively high levels of ultraviolet (UV)-B radiation. To gain first insights into how Pachycladon copes with UV-B stress, we sequenced its genome and compared the UV-B tolerance of two Pachycladon accessions with those of two A. thaliana accessions from different altitudes. Results A high-quality draft genome of P. cheesemanii was assembled with a high percentage of conserved single-copy plant orthologs. Synteny analysis with genomes from other species of the Brassicaceae family found a close phylogenetic relationship of P. cheesemanii with Boechera stricta from Brassicaceae lineage I. While UV-B radiation caused a greater growth reduction in the A. thaliana accessions than in the P. cheesemanii accessions, growth was not reduced in one P. cheesemanii accession. The homologues of A. thaliana UV-B radiation response genes were duplicated in P. cheesemanii, and an expression analysis of those genes indicated that the tolerance mechanism in P. cheesemanii appears to differ from that in A. thaliana. Conclusion Although the P. cheesemanii genome shows close similarity with that of A. thaliana, it appears to have evolved novel strategies allowing the plant to tolerate relatively high UV-B radiation.

Optimizing flowering time is crucial for maximizing crop productivity, but gaps remain in the knowledge of the mechanisms underpinning temperate legume flowering. Medicago, like winter annual Arabidopsis, accelerates flowering after exposure to extended cold (vernalization, V) followed by long-day (LD) photoperiods. In Arabidopsis, photoperiodic flowering is triggered through CO, a photoperiodic switch that directly activates the FT gene encoding a mobile florigen and potent activator of flowering. In Arabidopsis, several CYCLING DOF FACTORs (CDFs), including AtCDF1, act redundantly to repress CO and thus FT expression, until their removal in LD by a blue-light-induced F-BOX1/GIGANTEA (FKF1/GI) complex. Medicago possesses a homolog of FT, MtFTa1, which acts as a strong activator of flowering. However, the regulation of MtFTa1 does not appear to involve a CO-like gene. Nevertheless, work in pea suggests that CDFs may still regulate flowering time in temperate legumes. Here, we analyze the function of Medicago MtCDF genes with a focus on MtCDFd1_1 in flowering time and development. MtCDFd1_1 causes strong delays to flowering when overexpressed in Arabidopsis and shows a cyclical diurnal expression in Medicago with peak expression at dawn, consistent with AtCDF genes like AtCDF1. However, MtCDFd1_1 lacks predicted GI or FKF1 binding domains, indicating possible differences in its regulation from AtCDF1. In Arabidopsis, CDFs act in a redundant manner, and the same is likely true of temperate legumes as no flowering time phenotypes were observed when MtCDFd1_1 or other MtCDFs were knocked out in Medicago Tnt1 lines. Nevertheless, overexpression of MtCDFd1_1 in Medicago plants resulted in late flowering relative to wild type in inductive vernalized long-day (VLD) conditions, but not in vernalized short days (VSDs), rendering them day neutral. Expression of MtCO-like genes was not affected in the transgenic lines, but LD-induced genes MtFTa1, MtFTb1, MtFTb2, and MtSOC1a showed reduced expression. Plants carrying both the Mtfta1 mutation and 35S:MtCDFd1_1 flowered no later than the Mtfta1 plants. This indicates that 35S:MtCDFd1_1 likely influences flowering in VLD via repressive effects on MtFTa1 expression. Overall, our study implicates MtCDF genes in photoperiodic regulation in Medicago by working redundantly to repress FT-like genes, particularly MtFTa1, but in a CO-independent manner, indicating differences from the Arabidopsis model.

Optimizing flowering time is crucial for maximizing crop productivity, but gaps remain in the knowledge of the mechanisms underpinning temperate legume flowering. Medicago, like winter annual Arabidopsis, accelerates flowering after exposure to extended cold (vernalization, V) followed by long-day (LD) photoperiods. In Arabidopsis, photoperiodic flowering is triggered through CO, a photoperiodic switch that directly activates the FT gene encoding a mobile florigen and potent activator of flowering. In Arabidopsis, several CYCLING DOF FACTORs (CDFs), including AtCDF1, act redundantly to repress CO and thus FT expression, until their removal in LD by a blue-light-induced F-BOX1/GIGANTEA (FKF1/GI) complex. Medicago possesses a homolog of FT, MtFTa1, which acts as a strong activator of flowering. However, the regulation of MtFTa1 does not appear to involve a CO-like gene. Nevertheless, work in pea suggests that CDFs may still regulate flowering time in temperate legumes. Here, we analyze the function of Medicago MtCDF genes with a focus on MtCDFd1_1 in flowering time and development. MtCDFd1_1 causes strong delays to flowering when overexpressed in Arabidopsis and shows a cyclical diurnal expression in Medicago with peak expression at dawn, consistent with AtCDF genes like AtCDF1. However, MtCDFd1_1 lacks predicted GI or FKF1 binding domains, indicating possible differences in its regulation from AtCDF1. In Arabidopsis, CDFs act in a redundant manner, and the same is likely true of temperate legumes as no flowering time phenotypes were observed when MtCDFd1_1 or other MtCDFs were knocked out in Medicago Tnt1 lines. Nevertheless, overexpression of MtCDFd1_1 in Medicago plants resulted in late flowering relative to wild type in inductive vernalized long-day (VLD) conditions, but not in vernalized short days (VSDs), rendering them day neutral. Expression of MtCO-like genes was not affected in the transgenic lines, but LD-induced genes MtFTa1, MtFTb1, MtFTb2, and MtSOC1a showed reduced expression. Plants carrying both the Mtfta1 mutation and 35S:MtCDFd1_1 flowered no later than the Mtfta1 plants. This indicates that 35S:MtCDFd1_1 likely influences flowering in VLD via repressive effects on MtFTa1 expression. Overall, our study implicates MtCDF genes in photoperiodic regulation in Medicago by working redundantly to repress FT-like genes, particularly MtFTa1, but in a CO-independent manner, indicating differences from the Arabidopsis model.

The great hunt for florigen, the universal, long distance flowering regulator proposed by Chailakhan in the 1930s, resulted in the discovery a decade ago that FT-like proteins fulfilled the predictions for florigen. They are small (∼175 amino acids), globular, phosphatidylethanolamine-binding (PEBP) proteins, phloem-expressed, graft-transmissible and able to move to the shoot apex to act as potent stimulators of flowering in many plants. Genes that regulate Arabidopsis FT protein movement and some features of Arabidopsis FT protein that make it an effective florigen have recently been identified. Although floral promotion via graft transmission of FT has not been demonstrated in trees, FT-like genes have been successfully applied to reducing the long juvenile (pre-flowering) phase of many trees enabling fast track breeding.

Optimising the timing of flowering contributes to successful sexual reproduction and yield in agricultural plants. FLOWERING LOCUS T (FT) genes, first identified in Arabidopsis thaliana (Arabidopsis), promote flowering universally, but the upstream flowering regulatory pathways can differ markedly among plants. Flowering in the model legume, Medicago truncatula (Medicago) is accelerated by winter cold (vernalisation) followed by long day (LD) photoperiods leading to elevated expression of the floral activator, FT-like gene FTa1. However, Medicago, like some other plants, lacks the activator CONSTANS (CO) and the repressor FLOWERING LOCUS C (FLC) genes which directly regulate FT and are key to LD and vernalisation responses in Arabidopsis. Conversely, Medicago has a VERNALISATION2-LIKE VEFS-box gene (MtVRN2). In Arabidopsis AtVRN2 is a key member of a Polycomb complex involved in stable repression of Arabidopsis FLC after vernalisation. VRN2-like genes have been identified in other eudicot plants, but their function has never been reported. We show that Mtvrn2 mutants bypass the need for vernalisation for early flowering in LD conditions in Medicago. Investigation of the underlying mechanism by transcriptome analysis reveals that Mtvrn2 mutants precociously express FTa1 and other suites of genes including floral homeotic genes. Double-mutant analysis indicates that early flowering is dependent on functional FTa1. The broad significance of our study is that we have demonstrated a function for a VRN2-like VEFS gene beyond the Brassicaceae. In particular, MtVRN2 represses the transition to flowering in Medicago by regulating the onset of expression of the potent floral activator, FTa1.

Photoperiodic flowering aligns plant reproduction to favourable seasons of the year to maximise successful production of seeds and grains. However understanding of this process in the temperate legumes of the Fabaceae family, which are important both agriculturally and ecologically, is incomplete. Previous work in the reference legume Medicago truncatula has shown that the FT-like gene MtFTa1 is a potent floral activator. While MtFTa1 is upregulated by long-day photoperiods (LD) and vernalisation, the molecular basis of this is unknown as functional homologues of key regulatory genes present in other species, notably CONSTANS in A. thaliana, have not been identified. In LD MtFTa1 maintains a near constant diurnal pattern of expression unlike its homologue FT in A. thaliana, which has a notable peak in expression at dusk. This suggests a different manner of regulation. Furthermore, M. truncatula possesses other FT-like genes such as two LD induced MtFTb genes which may also act in the regulation of flowering time. MtFTb genes have a diurnal pattern of expression with peaks at both four and sixteen hours after dawn. This study utilises RNA-Seq to analyse the transcriptome of M. truncatula leaves to identify genes which may regulate or be co-expressed with these FT-like genes following a shift from short-day photoperiods to inductive long-days. Specifically this study focuses on the first four hours of the day in the young leaves, which coincides with the first diurnal peak of the FTb genes. Following differential expression analysis at each timepoint, genes which alter their pattern of expression are distinguished from those which just alter their magnitude of expression (and those that do neither). It goes on to categorise these genes into groups with similar patterns of expression using c-means clustering and identifies a number of potential candidate photoperiod flowering time genes for future studies to consider.

Medicago flowering, like that of Arabidopsis, is promoted by vernalization and long days, but alternative mechanisms are predicted because Medicago lacks the key regulators CO and FLC. Three Medicago SOC1-like genes, including MtSOC1a, were previously implicated in flowering control, but no legume soc1 mutants with altered flowering were reported. Here, reverse transciption-quantitative PCR (RT-qPCR) indicated that the timing and magnitude of MtSOC1a expression was regulated by the flowering promoter FTa1, while in situ hybridization indicated that MtSOC1a expression increased in the shoot apical meristem during the floral transition. A Mtsoc1a mutant showed delayed flowering and short primary stems. Overexpression of MtSOC1a partially rescued the flowering of Mtsoc1a, but caused a dramatic increase in primary stem height, well before the transition to flowering. Internode cell length correlated with stem height, indicating that MtSOC1a promotes cell elongation in the primary stem. However, application of gibberellin (GA3) caused stem elongation in both the wild type and Mtsoc1a, indicating that the mutant was not defective in gibberellin responsiveness. These results indicate that MtSOC1a may function as a floral integrator gene and promotes primary stem elongation. Overall, this study suggests that apart from some conservation with the Arabidopsis flowering network, MtSOC1a has a novel role in regulating aspects of shoot architecture.

Lolium perenne (ryegrass) is a perennial forage grass of worldwide importance. It is a temperate climate grass and flowering is induced by long day photoperiods. The agronomic productivity of ryegrass is strongly influenced by flowering time, but less is known about ryegrass flowering time regulators in other temperate Poaceae like wheat, barley and the model grass Brachypodium. The GIGANTEA (GI) gene was first identified in Arabidopsis and is an important regulator of photoperiodic flowering in angiosperms. GI usually exists as a conserved, single-copy gene. However, recently, genome sequencing has revealed that some plants, including the tropical grass maize, carry more than one full-length copy of GI. Here we describe the isolation and characterisation of the ryegrass L. perenne GIGANTEA gene (LpGI) gene. Comparative genomic analysis indicates that LpGI gene structure is very well conserved overall with GI genes from monocots and eudicots. LpGI protein clusters with GI proteins from other temperate grasses and is most closely related to the GI protein from meadow fescue. Genetic mapping locates LpGI to a chromosomal region that is syntenic with rice and Brachypodium GI. Our functional characterisation shows that LpGI shows a diurnal pattern of expression, continues to oscillate under constant light and adjusts its phase in response to changes in day length as seen in other plants. Constitutive expression of LpGI completely rescues the Arabidopsis gigantea-3 allele (gi-3) mutation, confirming that the isolated ryegrass gene is fully functional. Taken together, it is highly likely that LpGI is orthologous to Arabidopsis GI and involved in photoperiodic flowering time control in ryegrass.

* Article free to read on publisher website. Abstract Apples are rich in polyphenols, which provide antioxidant properties, mediation of cellular processes such as inflammation, and modulation of gut microbiota. In this study we compared genetically engineered apples with increased flavonoids [myeloblastis transcription factor 10 (MYB10)] with nontransformed apples from the same genotype, "Royal Gala" (RG), and a control diet with no apple. Compared with the RG diet, the MYB10 diet contained elevated concentrations of the flavonoid subclasses anthocyanins, flavanol monomers (epicatechin) and oligomers (procyanidin B2), and flavonols (quercetin glycosides), but other plant secondary metabolites were largely unaltered. We used these apples to investigate the effects of dietary flavonoids on inflammation and gut microbiota in 2 mouse feeding trials. In trial 1, male mice were fed a control diet or diets supplemented with 20% MYB10 apple flesh and peel (MYB-FP) or RG apple flesh and peel (RG-FP) for 7 d. In trial 2, male mice were fed MYB-FP or RG-FP diets or diets supplemented with 20% MYB10 apple flesh or RG apple flesh for 7 or 21 d. In trial 1, the transcription levels of inflammation-linked genes in mice showed decreases of >2-fold for interleukin-2 receptor (Il2rb), chemokine receptor 2 (Ccr2), chemokine ligand 10 (Cxcl10), and chemokine receptor 10 (Ccr10) at 7 d for the MYB-FP diet compared with the RG-FP diet (P

Photoperiodic flowering aligns plant reproduction to favourable seasons of the year to maximise successful production of seeds and grains. However understanding of this process in the temperate legumes of the Fabaceae family, which are important both agriculturally and ecologically, is incomplete. Previous work in the reference legume Medicago truncatula has shown that the FT-like gene MtFTa1 is a potent floral activator. While MtFTa1 is upregulated by long-day photoperiods (LD) and vernalisation, the molecular basis of this is unknown as functional homologues of key regulatory genes present in other species, notably CONSTANS in A. thaliana, have not been identified. In LD MtFTa1 maintains a near constant diurnal pattern of expression unlike its homologue FT in A. thaliana, which has a notable peak in expression at dusk. This suggests a different manner of regulation. Furthermore, M. truncatula possesses other FT-like genes such as two LD induced MtFTb genes which may also act in the regulation of flowering time. MtFTb genes have a diurnal pattern of expression with peaks at both four and sixteen hours after dawn. This study utilises RNA-Seq to analyse the transcriptome of M. truncatula leaves to identify genes which may regulate or be co-expressed with these FT-like genes following a shift from short-day photoperiods to inductive long-days. Specifically this study focuses on the first four hours of the day in the young leaves, which coincides with the first diurnal peak of the FTb genes. Following differential expression analysis at each timepoint, genes which alter their pattern of expression are distinguished from those which just alter their magnitude of expression (and those that do neither). It goes on to categorise these genes into groups with similar patterns of expression using c-means clustering and identifies a number of potential candidate photoperiod flowering time genes for future studies to consider.

SummaryBiological networks consist of a defined set of regulatory motifs. Subcellular compartmentalization of regulatory molecules can provide a further dimension in implementing regulatory motifs. However, spatial regulatory motifs and their roles in biological networks have rarely been explored. Here we show, using experimentation and mathematical modeling, that spatial segregation of GIGANTEA (GI), a critical component of plant circadian systems, into nuclear and cytosolic compartments leads to differential functions as positive and negative regulators of the circadian core gene, LHY, forming an incoherent feedforward loop to regulate LHY. This regulatory motif formed by nucleocytoplasmic partitioning of GI confers, through the balanced operation of the nuclear and cytosolic GI, strong rhythmicity and robustness to external and internal noises to the circadian system. Our results show that spatial and functional segregation of a single molecule species into different cellular compartments provides a means for extending the regulatory capabilities of biological networks.

Free to read at publisher FLOWERING LOCUS T (FT) and CENTRORADIALIS (CEN) homologs have been implicated in regulation of growth, determinacy and flowering. The roles of kiwifruit FT and CEN were explored using a combination of expression analysis, protein interactions, response to temperature in high-chill and low-chill kiwifruit cultivars and ectopic expression in Arabidopsis and Actinidia. The expression and activity of FT was opposite from that of CEN and incorporated an interaction with a FLOWERING LOCUS D (FD)-like bZIP transcription factor. Accumulation of FT transcript was associated with plant maturity and particular stages of leaf, flower and fruit development, but could be detected irrespective of the flowering process and failed to induce precocious flowering in transgenic kiwifruit. Instead, transgenic plants demonstrated reduced growth and survival rate. Accumulation of FT transcript was detected in dormant buds and stem in response to winter chilling. In contrast, FD in buds was reduced by exposure to cold. CEN transcript accumulated in developing latent buds, but declined before the onset of dormancy and delayed flowering when ectopically expressed in kiwifruit. Our results suggest roles for FT, CEN and FD in integration of developmental and environmental cues that affect dormancy, budbreak and flowering in kiwifruit.

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.

FLOWERING LOCUS T (FT) genes encode proteins that function as the mobile floral signal, florigen. In this study, we characterized five FT-like genes from the model legume, Medicago (Medicago truncatula). The different FT genes showed distinct patterns of expression and responses to environmental cues. Three of the FT genes (MtFTa1, MtFTb1, and MtFTc) were able to complement the Arabidopsis (Arabidopsis thaliana) ft-1 mutant, suggesting that they are capable of functioning as florigen. MtFTa1 is the only one of the FT genes that is up-regulated by both long days (LDs) and vernalization, conditions that promote Medicago flowering, and transgenic Medicago plants overexpressing the MtFTa1 gene flowered very rapidly. The key role MtFTa1 plays in regulating flowering was demonstrated by the identification of fta1 mutants that flowered significantly later in all conditions examined. fta1 mutants do not respond to vernalization but are still responsive to LDs, indicating that the induction of flowering by prolonged cold acts solely through MtFTa1, whereas photoperiodic induction of flowering involves other genes, possibly MtFTb1, which is only expressed in leaves under LD conditions and therefore might contribute to the photoperiodic regulation of flowering. The role of the MtFTc gene is unclear, as the ftc mutants did not have any obvious flowering-time or other phenotypes. Overall, this work reveals the diversity of the regulation and function of the Medicago FT family.

Anthocyanin concentration is an important determinant of the colour of many fruits. In apple (Malus x domestica), centuries of breeding have produced numerous varieties in which levels of anthocyanin pigment vary widely and change in response to environmental and developmental stimuli. The apple fruit cortex is usually colourless, although germplasm does exist where the cortex is highly pigmented due to the accumulation of either anthocyanins or carotenoids. From studies in a diverse array of plant species, it is apparent that anthocyanin biosynthesis is controlled at the level of transcription. Here we report the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple compared with a white-fleshed cultivar. We also describe an apple MYB transcription factor, MdMYB10, that is similar in sequence to known anthocyanin regulators in other species. We further show that this transcription factor can induce anthocyanin accumulation in both heterologous and homologous systems, generating pigmented patches in transient assays in tobacco leaves and highly pigmented apple plants following stable transformation with constitutively expressed MdMYB10. Efficient induction of anthocyanin biosynthesis in transient assays by MdMYB10 was dependent on the co-expression of two distinct bHLH proteins from apple, MdbHLH3 and MdbHLH33. The strong correlation between the expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this transcription factor is responsible for controlling anthocyanin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdMYB10 expression concurrent with colour formation during development. Characterization of MdMYB10 has implications for the development of new varieties through classical breeding or a biotechnological approach.

The timing of the transition to flowering is carefully controlled by plants in order to optimise sexual reproduction and the ensuing production of seeds, grains and fruits. The genetic networks that regulate floral induction are best characterised in the temperate eudicot Arabidopsis in which the florigen gene FT plays a major role in promoting the transition to flowering. Legumes are an important plant group, but less is known about the regulation of their flowering time. In the model legume Medicago truncatula (Medicago), a temperate annual plant like Arabidopsis, flowering is induced by prolonged cold (vernalisation) followed by long day lengths (LD). Recent molecular-genetic experiments have revealed that a FT-like gene, MtFTa1, is a central regulator of flowering time in Medicago. Here, we characterize the three Medicago FRUITFULL (FUL) MADS transcription factors, MtFULa, MtFULb and MtFULc using phylogenetic analyses, gene expression profiling through developmental time courses, and functional analyses in transgenic plants. MtFULa and MtFULb have similarity in sequence and expression profiles under inductive environmental conditions during both vegetative and reproductive development while MtFULc is only up regulated in the apex after flowering in LD conditions. Sustained up regulation of MtFULs requires functional MtFTa1 but their transcript levels are not affected during cold treatment. Overexpression of MtFULa and MtFULb promotes flowering in transgenic Arabidopsis plants with an additional terminal flower phenotype on some 35S:MtFULb plants. An increase in transcript levels of the MtFULs was also observed in Medicago plants overexpressing MtFTa1. Our results suggest that the MtFULs are targets of MtFTa1. Overall, this work highlights the conserved functions of FUL-like genes in promoting flowering and other roles in plant development and thus contributes to our understanding of the genetic control of the flowering process in Medicago.

Carboxylesterases hydrolyze esters of short-chain fatty acids and have roles in animals ranging from signal transduction to xenobiotic detoxification. In plants, however, little is known of their roles. We have systematically mined the genome from the model plant Arabidopsis thaliana for carboxylesterase genes and studied their distribution in the genome and expression profile across a range of tissues. Twenty carboxylesterase genes (AtCXE) were identified. The AtCXE family shares conserved sequence motifs and secondary structure characteristics with carboxylesterases and other members of the larger alpha/beta hydrolase fold superfamily of enzymes. Phylogenetic analysis of the AtCXE genes together with other plant carboxylesterases distinguishes seven distinct clades, with an Arabidopsis thaliana gene represented in six of the seven clades. The AtCXE genes are widely distributed across the genome (present in four of five chromosomes), with the exception of three clusters of tandemly duplicated genes. Of the interchromosomal duplication events, two have been mediated through newly identified partial chromosomal duplication events that also include other genes surrounding the AtCXE loci. Eighteen of the 20 AtCXE genes are expressed over a broad range of tissues, while the remaining 2 (unrelated) genes are expressed only in the flowers and siliques. Finally, hypotheses for the functional roles of the AtCXE family members are presented based on the phylogenetic relationships with other plant carboxylesterases of known function, their expression profile, and knowledge of likely esterase substrates found in plants.

CONSTANS promotes flowering of Arabidopsis in response to long-day conditions. We show that CONSTANS is a member of an Arabidopsis gene family that comprises 16 other members. The CO-Like proteins encoded by these genes contain two segments of homology: a zinc finger containing region near their amino terminus and a CCT (CO, CO-Like, TOC1) domain near their carboxy terminus. Analysis of seven classical co mutant alleles demonstrated that the mutations all occur within either the zinc finger region or the CCT domain, confirming that the two regions of homology are important for CO function. The zinc fingers are most similar to those of B-boxes, which act as protein-protein interaction domains in several transcription factors described in animals. Segments of CO protein containing the CCT domain localize GFP to the nucleus, but one mutation that affects the CCT domain delays flowering without affecting the nuclear localization function, suggesting that this domain has additional functions. All eight co alleles, including one recovered by pollen irradiation in which DNA encoding both B-boxes is deleted, are shown to be semidominant. This dominance appears to be largely due to a reduction in CO dosage in the heterozygous plants. However, some alleles may also actively delay flowering, because overexpression from the CaMV 35S promoter of the co-3 allele, that has a mutation in the second B-box, delayed flowering of wild-type plants. The significance of these observations for the role of CO in the control of flowering time is discussed.

Multigene families are common in higher organisms. However, due to the close similarities between members, it is often difficult to assess the individual contribution of each gene to the overall expression of the family. In Arabidopsis thaliana, there are four genes encoding the small subunits (SSU) of ribulose-1.5-bisphosphate carboxylase oxygenase (rubisco) whose nucleotide sequences are up to 98.4% identical. In order to overcome the technical limitations associated with gene-specific probes (or primers) commonly used in existing methods, we developed a new gene expression assay based on the RACE (rapid amplification of cDNA ends) technique with a single pair of primers. With this RACE gene expression assay, we were able to determine the relative transcript levels between four Arabidopsis SSU genes. We found that the relative SSU gene expression differed significantly between plants grown at different temperatures. Our observation raises the possibility that an adaptation of rubisco to the environment may be achieved through the specific synthesis of the SSU proteins, which is determined by the relative expression levels between the SSU genes.

Differential display was used to isolate genes differentially expressed early in fruit development of apple (Malus domestica Borkh.). This approach resulted in the isolation of MDH1, a homeobox gene with a homeodomain similar to that of BELL1 (BEL1), which is involved in regulation of ovule development in Arabidopsis. However, outside the homeodomain MDH1 is quite different from BEL1. In apple, MDH1 mRNA was predominantly found in flowers, expanding leaves and expanding fruit. In pre-anthesis flowers, in situ hybridization showed that MDH1 mRNA accumulated in ovules. To further investigate the function of this new homeobox gene, MDH1 was transformed into Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. The transgenic Arabidopsis plants showed dwarfing, reduced fertility and changes in carpel and fruit (silique) shape. The size and shape of the cells in the transgenic fruit was irregular. Both the transgenic phenotypes in Arabidopsis and the expression pattern of this gene in apple are consistent with the idea that MDH1 is likely to play an important role in control of plant fertility.

The dominant late elongated hypocotyl (lhy) mutation of Arabidopsis disrupted circadian clock regulation of gene expression and leaf movements and caused flowering to occur independently of photoperiod. LHY was shown to encode a MYB DNA-binding protein. In wild-type plants, the LHY mRNA showed a circadian pattern of expression with a peak around dawn but in the mutant was expressed constantly at high levels. Increased LHY expression from a transgene caused the endogenous gene to be expressed at a constant level, suggesting that LHY was part of a feedback circuit that regulated its own expression. Thus, constant expression of LHY disrupts several distinct circadian rhythms in Arabidopsis, and LHY may be closely associated with the central oscillator of the circadian clock.

The MADS-domain transcription factor SHORT VEGETATIVE PHASE plays a key role as a repressor of the transition to flowering and as a regulator of early floral development in Arabidopsis thaliana (Arabidopsis). However, no flowering-time repressors have been functionally identified in the model legume Medicago truncatula (Medicago). In this study, phylogenetic analysis of two closely-related MtSVP-like sequences, MtSVP1 and MtSVP2, showed that their predicted proteins clustered together within the eudicot SVP clade. To determine if the MtSVP-like genes have a role in flowering, they were functionally characterized in Medicago and Arabidopsis. Transcripts of both MtSVP genes were abundant and broadly expressed in vegetative tissues but were detected at much lower levels in flowers in Medicago. Over-expression of the MtSVP genes in Arabidopsis resulted in delayed flowering and flowers with many abnormal phenotypes such as leafy sepals, changes to floral organ number and longer pedicels than the wild type. By contrast, in transgenic Medicago, over-expression of MtSVP1 resulted in alterations to flower development, but did not alter flowering time, suggesting that MtSVP1 may not function to repress the transition to flowering in Medicago.

To extend our understanding of flowering time control in eudicots, we screened for mutants in the model legume Medicago truncatula (Medicago). We identified an early flowering mutant, spring1, in a T-DNA mutant screen, but spring1 was not tagged and was deemed a somaclonal mutant. We backcrossed the mutant to wild type R108. The F1 plants and the majority of F2 plants were early flowering like spring1, strongly indicating that spring1 conferred monogenic, dominant early flowering. We hypothesized that the spring1 phenotype resulted from over expression of an activator of flowering. Previously, a major QTL for flowering time in different Medicago accessions was located to an interval on chromosome 7 with six candidate flowering- time activators, including a CONSTANS gene, MtCO, and three FLOWERING LOCUS T (FT) genes. Hence we embarked upon linkage mapping using 29 markers from the MtCO/FT region on chromosome 7 on two populations developed by crossing spring1 with Jester. Spring1 mapped to an interval of ∼0.5 Mb on chromosome 7 that excluded MtCO, but contained 78 genes, including the three FT genes. Of these FT genes, only FTa1 was up-regulated in spring1 plants. We then investigated global gene expression in spring1 and R108 by microarray analysis. Overall, they had highly similar gene expression and apart from FTa1, no genes in the mapping interval were differentially expressed. Two MADS transcription factor genes, FRUITFULLb (FULb) and SUPPRESSOR OF OVER EXPRESSION OF CONSTANS1a (SOC1a), that were up-regulated in spring1, were also up-regulated in transgenic Medicago over-expressing FTa1. This suggested that their differential expression in spring1 resulted from the increased abundance of FTa1. A 6255 bp genomic FTa1 fragment, including the complete 5′ region, was sequenced, but no changes were observed indicating that the spring1 mutation is not a DNA sequence difference in the FTa1 promoter or introns.

The model dicotyledonous plant, Arabidopsis thaliana, is closely related to Brassica crop species. It is intended that information concerning the genetic control of basic biological processes in Arabidopsis will be transferable to other species. Genome collinearity and its potential to facilitate the identification of candidate genes in Arabidopsis homologous to genes controlling important agronomic traits in Brassica was investigated. Genetic mapping in B. nigra identified two loci influencing flowering time (FT), with loci on linkage groups 2 and 8 explaining 53% and 12% of the total variation in FT, respectively. The CO gene exerts an important control over FT in A. thaliana, and B. nigra homologues of CO probably also play an important role in regulating FT. B. nigra homologues of CO were identified on linkage groups 2 and 8, the homologue on group 2 was coincident with the major locus controlling FT while the homologue on group 8 was within the 90% confidence interval of the weaker FT gene. The CO homologue on group 2 exhibits abundant allelic variation suggesting that it naturally controls a wide range of flowering times. Fine-scale A. thaliana/B. nigra comparative mapping demonstrated short-range collinearity between the genomes of Arabidopsis and Brassica. Eleven DNA fragments spaced over a 1.5 Mb contig in A. thaliana were used as RFLP probes in B. nigra. Three collinear representations of the A. thaliana contig were identified in B. nigra, with one interrupted by a large chromosomal inversion. Collinearity over this range will allow the resources generated by the Arabidopsis genome project to facilitate map-based cloning in Brassica crops.

The vegetative and reproductive (flowering) phases of Arabidopsis development are clearly separated. The onset of flowering is promoted by long photoperiods, but the constans (co) mutant flowers later than wild type under these conditions. The CO gene was isolated, and two zinc fingers that show a similar spacing of cysteines, but little direct homology, to members of the GATA1 family were identified in the amino acid sequence. co mutations were shown to affect amino acids that are conserved in both fingers. Some transgenic plants containing extra copies of CO flowered earlier than wild type, suggesting that CO activity is limiting on flowering time. Double mutants were constructed containing co and mutations affecting gibberellic acid responses, meristem identity, or phytochrome function, and their phenotypes suggested a model for the role of CO in promoting flowering.

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 signalling system contributes to the unique plant architectures of different species is yet to be determined. We examined petunia orthologues of the Arabidopsis MAX1 and MAX2 genes to characterise their role in petunia architecture. A single orthologue 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 increase as leaves age. Nonetheless, this gene functionally complements the Arabidopsis max2 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 the development series. Alterations to the expression of these genes over time, or in different regions of the plant, may influence the branching growth habit of the plant. Alterations to strigolactone production and/or sensitivity could allow both subtle and dramatic changes to branching within and between species.

YAC clones carrying repeated DNA sequences from the Arabidopsis thaliana genome have been characterized in two widely used Arabidopsis YAC libraries, the EG library and the EW library. Ribosomal, chloroplast and the paracentromeric repeat sequences are differentially represented in the two libraries. The coordinates of YAC clones hybridizing to these sequences are given. A high proportion of EG YAC clones were classified as containing chimaeric inserts because individual clones carried unique sequences and repetitive sequences originating from different locations in the genome. None of the EW YAC clones analysed were chimaeric in this way. YAC clones carrying tandemly repeated sequences, such as the paracentromeric or rDNA sequences, exhibited a high degree of instability. These observations need to be taken into account when using these libraries in the development of a physical map of the Arabidopsis genome and in chromosome walking experiments.

Background The ability to induce flowering on demand is of significant biotechnological interest. FT protein has been recently identified as an important component of the mobile flowering hormone, florigen, whose function is conserved across the plant kingdom. We therefore focused on manipulation of both endogenous and heterologous FT genes to develop a floral induction system where flowering would be inhibited until it was induced on demand. The concept was tested in the model plant Arabidopsis thaliana (Arabidopsis). Results Our starting point was plants with strongly delayed flowering due to silencing of FT with an artificial microRNA directed at FT (amiR-FT) [1]. First, we showed that constitutive expression of a heterologous FT gene (FTa1), from the model legume Medicago truncatula, (Medicago) was able to rescue the amiR-FT late-flowering phenotype. In order to induce flowering in a controlled way, the FTa1 gene was then expressed under the control of an alcohol-inducible promoter in the late flowering amiR-FT plants. Upon exposure to ethanol, FTa1 was rapidly up regulated and this resulted in the synchronous induction of flowering. Conclusions We have thus demonstrated a controlled-inducible flowering system using a novel combination of endogenous and heterologous FT genes. The universal florigenic nature of FT suggests that this type of system should be applicable to crops of economic value where flowering control is desirable.

The co mutation of Arabidopsis thaliana causes a late-flowering phenotype that is insensitive to day-length. The mutation was mapped previously to the upper arm of chromosome 5, approximately 1.6 cM from the chalcone synthase gene (CHS). We were provided with five yeast artificial chromosome (YAC) libraries and used these to perform a chromosome walk from CHS to the CO gene. In this paper we report the isolation of 1700 kb of contiguous Arabidopsis DNA, which represents approximately 1%-2% of the genome, inserted in YACs. This required the detailed analysis of 67 YACs, from which 87 end probes were isolated and examined in hybridisation experiments. This analysis showed that approximately 40% of the YACs presented problems in chromosome walking experiments because they contained repetitive sequence at one of their termini, were chimaeric or because part of the plant DNA was deleted. DNA fragments isolated from YACs were used as restriction fragment length polymorphism (RFLP) markers to localize CO to a 300 kb region within the cloned DNA. We compare the physical distance between CHS and CO with the genetic distance and find that in this region 1 cM is equivalent to approximately 200 kb.

The Arabidopsis thaliana ( Arabidopsis ) GIGANTEA ( GI ) gene is a central component of the photoperiodic flowering pathway. While it has been 40 years since the first mutant alleles of GI were described much is still unknown about the molecular mechanism of GI action. To investigate the biochemistry and domain organisation (and ultimately to give a greater understanding of the role of GI in floral induction), it is first necessary to produce significant quantities of purified protein. Soluble affinity-tagged full-length GI was expressed in Escherichia coli ( E. coli ) and was stabilised by the addition of the detergent n -dodecyl-β- d -maltoside (DDM) to storage and purification buffers. Stabilised GI was purified using a variety of chromatographic methods, and characterised using a selection of biochemical techniques including circular dichroism, and dynamic light scattering. This showed that purified GI contained secondary structure, but was polydisperse in solution. Electron microscopy suggests a possible tetramer arrangement of GI. Limited proteolytic digests and mass spectrometry were used to identify potential GI domains. This led to the identification of a predicted 46 kDa amino-terminal GI domain. GI was also expressed in Sf9 insect cells using the baculovirus expression system. GI produced via this route gave insoluble protein.