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Abstract 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.

Optimized flowering time is an important trait that ensures successful plant adaptation and crop productivity. SOC1-like genes encode MADS transcription factors, which are known to play important roles in flowering control in many plants. This includes the best-characterized eudicot model Arabidopsis thaliana (Arabidopsis), where SOC1 promotes flowering and functions as a floral integrator gene integrating signals from different flowering-time regulatory pathways. Medicago truncatula (Medicago) is a temperate reference legume with strong genomic and genetic resources used to study flowering pathways in legumes. Interestingly, despite responding to similar floral-inductive cues of extended cold (vernalization) followed by warm long days (VLD), such as in winter annual Arabidopsis, Medicago lacks FLC and CO which are key regulators of flowering in Arabidopsis. Unlike Arabidopsis with one SOC1 gene, multiple gene duplication events have given rise to three MtSOC1 paralogs within the Medicago genus in legumes: one Fabaceae group A SOC1 gene, MtSOC1a, and two tandemly repeated Fabaceae group B SOC1 genes, MtSOC1b and MtSOC1c. Previously, we showed that MtSOC1a has unique functions in floral promotion in Medicago. The Mtsoc1a Tnt1 retroelement insertion single mutant showed moderately delayed flowering in long- and short-day photoperiods, with and without prior vernalization, compared to the wild-type. In contrast, Mtsoc1b Tnt1 single mutants did not have altered flowering time or flower development, indicating that it was redundant in an otherwise wild-type background. Here, we describe the generation of Mtsoc1a Mtsoc1b Mtsoc1c triple mutant lines using CRISPR-Cas9 gene editing. We studied two independent triple mutant lines that segregated plants that did not flower and were bushy under floral inductive VLD. Genotyping indicated that these non-flowering plants were homozygous for the predicted strong mutant alleles of the three MtSOC1 genes. Gene expression analyses using RNA-seq and RT-qPCR indicated that these plants remained vegetative. Overall, the non-flowering triple mutants were dramatically different from the single Mtsoc1a mutant and the Arabidopsis soc1 mutant; implicating multiple MtSOC1 genes in critical overlapping roles in the transition to flowering in Medicago.

Anthocyanins are a major group of red to blue spectrum plant pigments with many consumer health benefits. Anthocyanins are derived from the flavonoid pathway and diversified by glycosylation and methylation, involving the concerted action of specific enzymes. Blueberry and bilberry (Vaccinium spp.) are regarded as ‘superfruits’ owing to their high content of flavonoids, especially anthocyanins. While ripening-related anthocyanin production in bilberry (V. myrtillus) and blueberry (V. corymbosum) is regulated by the transcriptional activator MYBA1, the role of specific structural genes in determining the concentration and composition of anthocyanins has not been functionally elucidated. We isolated three candidate genes, CHALCONE SYNTHASE (VmCHS1), ANTHOCYANIDIN SYNTHASE (VmANS) and UDP-GLUCOSE : FLAVONOID-3-O-GLYCOSYLTRANSFERASE (VcUFGT2), from Vaccinium, which were predominantly expressed in pigmented fruit skin tissue and showed high homology between bilberry and blueberry. Agrobacterium-mediated transient expression of Nicotiana benthamiana showed that overexpression of VcMYBA1 in combination with VmANS significantly increased anthocyanin concentration (3-fold). Overexpression of VmCHS1 showed no effect above that induced by VcMYBA1, while VcUFGT2 modulated anthocyanin composition to produce delphinidin-3-galactosylrhamnoside, not naturally produced in tobacco. In strawberry (Fragaria × ananassa), combined transient overexpression of VcUFGT2 with a FLAVONOID 3´,5´-HYDROXYLASE from kiwifruit (Actinidia melanandra) modulated the anthocyanin profile to include galactosides and arabinosides of delphinidin and cyanidin, major anthocyanins in blueberry and bilberry. These findings provide insight into the role of the final steps of biosynthesis in modulating anthocyanin production in Vaccinium and may contribute to the targeted breeding of new cultivars with improved nutritional properties.

Abstract Background Flowering time is an important trait for productivity in legumes, which include many food and fodder plants. Medicago truncatula (Medicago) is a model temperate legume used to study flowering time pathways. Like Arabidopsis thaliana (Arabidopsis), its flowering is promoted by extended periods of cold (vernalization, V), followed by warm long day (LD) photoperiods. However, Arabidopsis flowering-time genes such as the FLOWERING LOCUS C (FLC)/ MADS AFFECTING FLOWERING (MAF) clade are missing and CONSTANS-LIKE (CO-LIKE) genes do not appear to have a role in Medicago or Pisum sativum (pea). Another photoperiodic regulator, the red/far red photoreceptor PHYTOCHROME A (PHYA), promotes Arabidopsis flowering by stabilizing the CO protein in LD. Interestingly, despite the absence of CO-LIKE function in pea, PsPHYA plays a key role in promoting LD photoperiodic flowering and plant architecture. Medicago has one homolog of PHYA, MtPHYA, but its function is not known. Results Genetic analysis of two MtPHYA Tnt1 insertion mutant alleles indicates that MtPHYA has an important role in promoting Medicago flowering and primary stem elongation in VLD and LD and in perception of far-red wavelengths in seedlings. MtPHYA positively regulates the expression of MtE1-like (MtE1L), a homologue of an important legume-specific flowering time gene, E1 in soybean and other Medicago LD-regulated flowering-time gene homologues, including the three FLOWERING LOCUS T-LIKE (FT-LIKE) genes, MtFTa1, MtFTb1 and MtFTb2 and the two FRUITFULL-LIKE (FUL-LIKE) genes MtFULa and MtFULb. MtPHYA also modulates the expression of the circadian clock genes, GIGANTEA (GI) and TIMING OF CAB EXPRESSION 1a (TOC1a). Genetic analyses indicate that Mtphya-1 Mte1l double mutants flowered at the same time as the single mutants. However, Mtphya-1 Mtfta1 double mutants had a weak additive effect in delaying flowering and in reduction of primary axis lengths beyond what was conferred by either of the single mutants. Conclusion MtPHYA has an important role in LD photoperiodic control of flowering, plant architecture and seedling de-etiolation under far-red wavelengths in Medicago. It promotes the expression of LD-induced flowering time genes and modulates clock-related genes. In addition to MtFTa1, MtPHYA likely regulates other targets during LD floral induction in Medicago.

Abstract 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.

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.

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.

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.

Abstract Kiwifruit (Actinidia chinensis) has three FLOWERING LOCUS T (FT) genes, AcFT, AcFT1, and AcFT2, with differential expression and potentially divergent roles. AcFT was previously shown to be expressed in source leaves and induced in dormant buds by winter chilling. Here, we show that AcFT promotes flowering in A. chinensis, despite a short sequence insertion not present in other FT‐like genes. A 3.5‐kb AcFT promoter region contained all the regulatory elements required to mediate vascular expression in transgenic Arabidopsis thaliana (Arabidopsis). The promoter activation was initially confined to the veins in the distal end of the leaf, before extending to the veins in the base of the leaf, and was detected in inductive and noninductive photoperiods. The 3‐kb and 2.7‐kb promoter regions of AcFT1 and AcFT2, respectively, demonstrated different activation patterns in Arabidopsis, corresponding to differential expression in kiwifruit. Expression of AcFT cDNA from the AcFT promoter was capable to induce early flowering in transgenic Arabidopsis in noninductive photoperiods. Further, expression of AcFT cDNA fused to the green fluorescent protein was detected in the vasculature and was also capable to advance flowering in noninductive photoperiods. Taken together, these studies implicate AcFT in regulation of kiwifruit flowering time and as a candidate for kiwifruit florigen.

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.

Four male cone-specific promoters were isolated from the genome of Pinus radiata D. Don, fused to the ß-glucuronidase (GUS) reporter gene and analysed in the heterologous host Arabidopsis thaliana (L.) Heynh. The temporal and spatial activities of the promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1 during seven anther developmental stages are described in detail. The two promoters PrMC2 and PrMALE1 confer an identical GUS expression pattern on Arabidopsis anthers. DNA sequence analysis of the PrMC2 and PrMALE1 promoters revealed an 88% sequence identity over 276 bp and divergence further upstream (<40% sequence identity). GUS expression driven by a 276-bp PrMALE1 promoter fragment showed the same pattern in Arabidopsis anthers as observed for the full-length PrMALE1 promoter. Within the 276-bp promoter fragment a region of high homology to a previously described 16-bp anther-box was identified. In gain-of-function experiments the putative PrMALE1 anther-box was fused upstream of a 90-bp CaMV 35S minimal promoter, as a single copy in the sense direction and as an inverted repeat. No GUS expression was conferred to Arabidopsis anthers by either of these two constructs. In a loss-of-function experiment a 226-bp PrMALE1 deletion construct, which did not contain the putative PrMALE1 anther-box, still maintained the originally observed PrMALE1 GUS expression pattern. Hence, gain-of-function as well as loss-of-function experiments consistently showed that the putative anther-box of the PrMALE1 promoter is non-functional in the Arabidopsis genetic background. For the analysis of the four full-length pine promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1, transformation vectors based on pCAMBIA2200 and pCAMBIA1302 were used. It will also be demonstrated in this article that sequences within the T-DNA borders of these vectors caused a characteristic histological background expression in Arabidopsis, with staining observed in vascular tissue of leaves, sepals, roots, filaments of stamens and in stems and pistils. [ABSTRACT FROM AUTHOR]