Mutations in AP22.65 accelerate flowering in Arabidopsis thaliana

Identification of the gene(s) responsible for flowering time in Arabidopsis has significant implications. We used the T-DNA insertion library of Arabidopsis thaliana to screen an early-flowering mutant that exhibits accelerated flowering under short-day conditions. AP22.65, a novel flowering-time gene in that species, was isolated and identified via genome-walking and bioinformatics analysis. The flowering time of AP22.65-complementing plants was similar to that of the Col-0 wild type (WT). Conversely, its overexpression delayed flowering. Consistent with this phenotype, expression of AP22.65 was decreased in the ap22.65-1 mutant, recovered in AP22.65-complementing plants, and increased in AP22.65-overexpressing plants. Compared with the WT, expression levels of critical genes in different flowering pathways, i.e., SPY, FLC, GI, CO, FT, and LFY, were down-regulated in loss-of-function mutants. Expression of AP22.65 was distributed in flowers, siliques, rosette leaves, and whole seedlings. Therefore, this gene may be a negative regulator of Arabidopsis flowering.     

Major Soybean Maturity Gene Haplotypes Revealed by SNPViz Analysis of 72 Sequenced Soybean Genomes

In this Genomics Era, vast amounts of next-generation sequencing data have become publicly available for multiple genomes across hundreds of species. Analyses of these large-scale datasets can become cumbersome, especially when comparing nucleotide polymorphisms across many samples within a dataset and among different datasets or organisms. To facilitate the exploration of allelic variation and diversity, we have developed and deployed an in-house computer software to categorize and visualize these haplotypes. The SNPViz software enables users to analyze region-specific haplotypes from single nucleotide polymorphism (SNP) datasets for different sequenced genomes. The examination of allelic variation and diversity of important soybean [Glycine max (L.) Merr.] flowering time and maturity genes may provide additional insight into flowering time regulation and enhance researchers'' ability to target soybean breeding for particular environments. For this study, we utilized two available soybean genomic datasets for a total of 72 soybean genotypes encompassing cultivars, landraces, and the wild species Glycine soja. The major soybean maturity genes E1, E2, E3, and E4 along with the Dt1 gene for plant growth architecture were analyzed in an effort to determine the number of major haplotypes for each gene, to evaluate the consistency of the haplotypes with characterized variant alleles, and to identify evidence of artificial selection. The results indicated classification of a small number of predominant haplogroups for each gene and important insights into possible allelic diversity for each gene within the context of known causative mutations. The software has both a stand-alone and web-based version and can be used to analyze other genes, examine additional soybean datasets, and view similar genome sequence and SNP datasets from other species.     

Heterologous Expression of Wheat VERNALIZATION 2 (TaVRN2) Gene in Arabidopsis Delays Flowering and Enhances Freezing Tolerance

The vernalization gene 2 (VRN2), is a major flowering repressor in temperate cereals that is regulated by low temperature and photoperiod. Here we show that the gene from Triticum aestivum (TaVRN2) is also regulated by salt, heat shock, dehydration, wounding and abscissic acid. Promoter analysis indicates that TaVRN2 regulatory region possesses all the specific responsive elements to these stresses. This suggests pleiotropic effects of TaVRN2 in wheat development and adaptability to the environment. To test if TaVRN2 can act as a flowering repressor in species different from the temperate cereals, the gene was ectopically expressed in the model plant Arabidopsis. Transgenic plants showed no alteration in morphology, but their flowering time was significantly delayed compared to controls plants, indicating that TaVRN2, although having no ortholog in Brassicaceae, can act as a flowering repressor in these species. To identify the possible mechanism by which TaVRN2 gene delays flowering in Arabidopsis, the expression level of several genes involved in flowering time regulation was determined. The analysis indicates that the late flowering of the 35S::TaVRN2 plants was associated with a complex pattern of expression of the major flowering control genes, FCA, FLC, FT, FVE and SOC1. This suggests that heterologous expression of TaVRN2 in Arabidopsis can delay flowering by modulating several floral inductive pathways. Furthermore, transgenic plants showed higher freezing tolerance, likely due to the accumulation of CBF2, CBF3 and the COR genes. Overall, our data suggests that TaVRN2 gene could modulate a common regulator of the two interacting pathways that regulate flowering time and the induction of cold tolerance. The results also demonstrate that TaVRN2 could be used to manipulate flowering time and improve cold tolerance in other species.     

PORPHOBILINOGEN DEAMINASE Deficiency Alters Vegetative and Reproductive Development and Causes Lesions in Arabidopsis

The Arabidopsis rugosa1 (rug1) mutant has irregularly shaped leaves and reduced growth. In the absence of pathogens, leaves of rug1 plants have spontaneous lesions reminiscent of those seen in lesion-mimic mutants; rug1 plants also express cytological and molecular markers associated with defence against pathogens. These rug1 phenotypes are made stronger by dark/light transitions. The rug1 mutant also has delayed flowering time, upregulation of the floral repressor FLOWERING LOCUS C (FLC) and downregulation of the flowering promoters FT and SOC1/AGL20. Vernalization suppresses the late flowering phenotype of rug1 by repressing FLC. Microarray analysis revealed that 280 nuclear genes are differentially expressed between rug1 and wild type; almost a quarter of these genes are involved in plant defence. In rug1, the auxin response is also affected and several auxin-responsive genes are downregulated. We identified the RUG1 gene by map-based cloning and found that it encodes porphobilinogen deaminase (PBGD), also known as hydroxymethylbilane synthase, an enzyme of the tetrapyrrole biosynthesis pathway, which produces chlorophyll, heme, siroheme and phytochromobilin in plants. PBGD activity is reduced in rug1 plants, which accumulate porphobilinogen. Our results indicate that Arabidopsis PBGD deficiency impairs the porphyrin pathway and triggers constitutive activation of plant defence mechanisms leading to leaf lesions and affecting vegetative and reproductive development.     

Reduction of the geomagnetic field delays Arabidopsis thaliana flowering time through downregulation of flowering‐related genes

Variations in magnetic field (MF) intensity are known to induce plant morphological and gene expression changes. In Arabidopsis thaliana Col‐0, near‐null magnetic field (NNMF, i.e., <100 nT MF) causes a delay in the transition to flowering, but the expression of genes involved in this response has been poorly studied. Here, we showed a time‐course quantitative analysis of the expression of both leaf (including clock genes, photoperiod pathway, GA20ox, SVP, and vernalization pathway) and floral meristem (including GA2ox, SOC1, AGL24, LFY, AP1, FD, and FLC) genes involved in the transition to flowering in A. thaliana under NNMF. NNMF induced a delayed flowering time and a significant reduction of leaf area index and flowering stem length, with respect to controls under geomagnetic field. Generation experiments (F₁‐ and F₂‐NNMF) showed retention of flowering delay. The quantitative expression (qPCR) of some A. thaliana genes expressed in leaves and floral meristem was studied during transition to flowering. In leaves and flowering meristem, NNMF caused an early downregulation of clock, photoperiod, gibberellin, and vernalization pathways and a later downregulation of TSF, AP1, and FLC. In the floral meristem, the downregulation of AP1, AGL24, FT, and FLC in early phases of floral development was accompanied by a downregulation of the gibberellin pathway. The progressive upregulation of AGL24 and AP1 was also correlated to the delayed flowering by NNMF. The flowering delay is associated with the strong downregulation of FT, FLC, and GA20ox in the floral meristem and FT, TSF, FLC, and GA20ox in leaves. Bioelectromagnetics. 39:361–374, 2018. © 2018 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.     

Overexpression of a Brassica rapa MADS-box gene, BrAGL20, induces early flowering time phenotypes in Brassica napus

BrAGL20 (SOC1) containing MADS box, a floral integrator gene, was introduced into Brassica napus cv. “Youngsan” by Agrobacterium-mediated transformation. Constitutively overexpressed BrAGL20 under the CaMV 35S promoter induced early flowering time compared to the wild-type. These phenotypes were stably inherited through generations T₂ and T₃, regardless of planting season. The expression of the floral meristem identity genes LFY and AP1 seemed to appear rapidly in the shoot apex region of transgenic plants showing the early flowering time phenotype. These results suggest that overexpression of BrAGL20 can significantly affect the flowering time of B. napus, and regulation of floral integrator gene expression could be applied for adaptation of crops to local environments and climate changes.     

Nucleotide diversity of the upstream region of the putative MADS-box gene controlling soybean maturity

MADS-box genes are involved in plant reproductive development. However, the role of gene nucleotide diversity in soybean flowering and maturity remains unknown. Therefore, in this study, the distribution of DNA polymorphisms in the putative MADS-box gene located near the quantitative trait loci (QTL) for flowering time and maturity was targeted for association analysis using Glycine max (cultivated soybean) and Glycine soja (wild soybean). Sixteen single nucleotide polymorphisms identified in the upstream region of the putative MADS-box gene around QTL Pod mat 13-7 and Fflr 4-2 on chromosome 7 were found to be highly associated with maturity in soybean. The genetic diversity between cultivated soybeans and the wild relative was comparable, although the early maturity group (EMG) was less diverse than the late maturity group (LMG) of the cultivated soybean. Population size changes of the MADS-box gene in this soybean germplasm appeared to result from non-random selection. A selective pressure seemed to act on this gene in the EMG, while the LMG and G. soja were in genetic equilibrium. Neutrality tests and the constructed neighbor-joining tree indicate that the EMG of G. max has experienced strong artificial selection for its domestication and genetic improvement.     

Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis

Previous studies have shown that ubiquitination plays important roles in plant abiotic stress responses. In the present study, the ubiquitin-conjugating enzyme gene GmUBC2, a homologue of yeast RAD6, was cloned from soybean and functionally characterized. GmUBC2 was expressed in all tissues in soybean and was up-regulated by drought and salt stress. Arabidopsis plants overexpressing GmUBC2 were more tolerant to salinity and drought stresses compared with the control plants. Through expression analyses of putative downstream genes in the transgenic plants, we found that the expression levels of two ion antiporter genes AtNHX1 and AtCLCa, a key gene involved in the biosynthesis of proline, AtP5CS, and the copper chaperone for superoxide dismutase gene AtCCS, were all increased significantly in the transgenic plants. These results suggest that GmUBC2 is involved in the regulation of ion homeostasis, osmolyte synthesis, and oxidative stress responses. Our results also suggest that modulation of the ubiquitination pathway could be an effective means of improving salt and drought tolerance in plants through genetic engineering.     

Homologs of APETALA1/FRUITFULL in Solanum plants

The MADS-box APETALA1 genes control plant transition to flowering and the floral morphogenesis proper. The experimental evidence of APETALA1 overexpression presumes that this class of genes can also directly affect time to flowering. We therefore cloned and compared homologs of APETALA1 class genes from potato (Solanum tuberosum cultivars adapted to long day conditions) and its wild relative Solanum demissum, a short-day subtropical species. The homologs isolated from these plants belong to the subclass FRUITFULL. The inconsiderable variations in the primary structure of these homologs cannot explain the diverse photoperiodic reactions of particular Solanum genotypes.     

Dt2 Is a Gain-of-Function MADS-Domain Factor Gene That Specifies Semideterminacy in Soybean

Similar to Arabidopsis thaliana, the wild soybeans (Glycine soja) and many cultivars exhibit indeterminate stem growth specified by the shoot identity gene Dt1, the functional counterpart of Arabidopsis TERMINAL FLOWER1 (TFL1). Mutations in TFL1 and Dt1 both result in the shoot apical meristem (SAM) switching from vegetative to reproductive state to initiate terminal flowering and thus produce determinate stems. A second soybean gene (Dt2) regulating stem growth was identified, which, in the presence of Dt1, produces semideterminate plants with terminal racemes similar to those observed in determinate plants. Here, we report positional cloning and characterization of Dt2, a dominant MADS domain factor gene classified into the APETALA1/SQUAMOSA (AP1/SQUA) subfamily that includes floral meristem (FM) identity genes AP1, FUL, and CAL in Arabidopsis. Unlike AP1, whose expression is limited to FMs in which the expression of TFL1 is repressed, Dt2 appears to repress the expression of Dt1 in the SAMs to promote early conversion of the SAMs into reproductive inflorescences. Given that Dt2 is not the gene most closely related to AP1 and that semideterminacy is rarely seen in wild soybeans, Dt2 appears to be a recent gain-of-function mutation, which has modified the genetic pathways determining the stem growth habit in soybean.     

A Quantitative and Dynamic Model of the Arabidopsis Flowering Time Gene Regulatory Network

Various environmental signals integrate into a network of floral regulatory genes leading to the final decision on when to flower. Although a wealth of qualitative knowledge is available on how flowering time genes regulate each other, only a few studies incorporated this knowledge into predictive models. Such models are invaluable as they enable to investigate how various types of inputs are combined to give a quantitative readout. To investigate the effect of gene expression disturbances on flowering time, we developed a dynamic model for the regulation of flowering time in Arabidopsis thaliana. Model parameters were estimated based on expression time-courses for relevant genes, and a consistent set of flowering times for plants of various genetic backgrounds. Validation was performed by predicting changes in expression level in mutant backgrounds and comparing these predictions with independent expression data, and by comparison of predicted and experimental flowering times for several double mutants. Remarkably, the model predicts that a disturbance in a particular gene has not necessarily the largest impact on directly connected genes. For example, the model predicts that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) mutation has a larger impact on APETALA1 (AP1), which is not directly regulated by SOC1, compared to its effect on LEAFY (LFY) which is under direct control of SOC1. This was confirmed by expression data. Another model prediction involves the importance of cooperativity in the regulation of APETALA1 (AP1) by LFY, a prediction supported by experimental evidence. Concluding, our model for flowering time gene regulation enables to address how different quantitative inputs are combined into one quantitative output, flowering time.