Early Flowering Mutant Research Articles

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

Induced mutations in circadian clock regulator Mat-a facilitated short-season adaptation and range extension in cultivated barley

Time to flowering has an important impact on yield and has been a key trait in the domestication of crop plants and the spread of agriculture. In 1961, the cultivar Mari (mat-a.8) was the very first induced early barley (Hordeum vulgare L.) mutant to be released into commercial production. Mari extended the range of two-row spring barley cultivation as a result of its photoperiod insensitivity. Since its release, Mari or its derivatives have been used extensively across the world to facilitate short-season adaptation and further geographic range extension. By exploiting an extended historical collection of early-flowering mutants of barley, we identified Praematurum-a (Mat-a), the gene responsible for this key adaptive phenotype, as a homolog of the Arabidopsis thaliana circadian clock regulator Early Flowering 3 (Elf3). We characterized 87 induced mat-a mutant lines and identified >20 different mat-a alleles that had clear mutations leading to a defective putative ELF3 protein. Expression analysis of HvElf3 and Gigantea in mutant and wild-type plants demonstrated that mat-a mutations disturb the flowering pathway, leading to the early phenotype. Alleles of Mat-a therefore have important and demonstrated breeding value in barley but probably also in many other day-length-sensitive crop plants, where they may tune adaptation to different geographic regions and climatic conditions, a critical issue in times of global warming.

The Arabidopsis CstF64-Like RSR1/ESP1 Protein Participates in Glucose Signaling and Flowering Time Control

Mechanisms for sensing and regulating metabolic processes at the cellular level are critical for the general physiology and development of living organisms. In higher plants, sugar signaling is crucial for adequate regulation of carbon and energy metabolism and affects virtually every aspect of development. Although many genes are regulated by sugar levels, little is known on how sugar levels are measured by plants. Several components of the sugar signaling network have been unraveled and demonstrated to have extensive overlap with hormone signaling networks. Here we describe the reduced sugar response1-1 (rsr1-1) mutant as a new early flowering mutant that displays decreased sensitivity to abscisic acid. Both hexokinase1 (HXK1)-dependent and glucose phosphorylation-independent signaling is reduced in rsr1-1. Map-based identification of the affected locus demonstrated that rsr1-1 carries a premature stop codon in the gene for a CstF64-like putative RNA processing factor, ESP1, which is involved in mRNA 3′-end formation. The identification of RSR1/ESP1 as a nuclear protein with a potential threonine phosphorylation site may explain the impact of protein phosphorylation cascades on sugar-dependent signal transduction. Additionally, RSR1/ESP1 may be a crucial factor in linking sugar signaling to the control of flowering time.

Identification of miRNAs and Their Target Genes Using Deep Sequencing and Degradome Analysis in Trifoliate Orange [Poncirus trifoliate (L.) Raf

To identify novel as well as conserved miRNAs in citrus, deep sequencing of small RNA library combined with microarray was performed in precocious trifoliate orange (an early flowering mutant of trifoliate orange, Poncirus trifoliata L. Raf.), resulting in the obtainment of a total of 114 conserved miRNAs belonging to 38 families and 155 novel miRNAs. The miRNA star sequences of 39 conserved miRNAs and 27 novel miRNAs were also discovered among newly identified miRNAs, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 172 and 149 genes were identified as targets of conserved miRNAs and novel miRNAs, respectively. GO and KEGG annotation revealed that high ranked miRNA-target genes were those implicated in biological and metabolic processes. To characterize those miRNAs expressed at the juvenile and adult development stages of citrus, further analysis on the expression profiles of these miRNAs through hybridizing the commercial microarray and real-time PCR was performed. The results revealed that some miRNAs were down-regulated at adult stage compared with juvenile stage. Detailed comparison of the expression patterns of some miRNAs and corresponding target genes revealed the negative correlation between them, while few of them are positively correlated.

Construction of RNAi vector for flower-related gene and verifications of the mutant in Arabidopsis thaliana

The timing of floral transition is tightly controlled by a combination of endogenous and environmental signals. One early flowering mutant plant was screened from Arabidopsis library of T-DNA insertion to accelerate flowering under short-day condition, and a related-gene EFS1 (AT4G36680.1) was isolated and identified as a novel flowering-time gene of Arabidopsis in our preliminary studies. To investigate the function and the specific mechanism of EFS1 in the flower process control, the RNAi expression vector containing EFS1 gene-specific sequences in the sense and antisense orientations was constructed and transferred into Arabidopsis by using the floral-dip method, with 11 transgenic plants obtained through hygromycin B screening and PCR assays. The results showed that the expression level of EFS1 in transgenic lines was significantly lower than that in wild type and efs1 mutant. The flowering time of the efs1 mutant and RNAi transgenic plants was much earlier than that of wild-type plants. This result further verified that the EFS1 gene played an important role in flowering, and its specific mechanisms need further study. These work provided a foundation to further regulatory mechanisms of EFS1 in the control of floral transition.

PtSVP, an SVP homolog from trifoliate orange (Poncirus trifoliata L. Raf.), shows seasonal periodicity of meristem determination and affects flower development in transgenic Arabidopsis and tobacco plants

A MADS-box gene was isolated using the suppressive subtractive hybridization library between early-flowering mutant and wild-type trifoliate orange (Poncirus trifoliata L. Raf.). This gene is highly homologous with Arabidopsis SHORT VEGETATIVE PHASE (SVP). Based on real-time PCR and in situ hybridization during bud differentiation, PtSVP was expressed intensively in dormant tissue and vegetative meristems. PtSVP transcripts were detected in apical meristems before floral transition, then down-regulated during the transition. PtSVP expression was higher in differentiated (flower primordium) than in undifferentiated cells (apical meristems). The PtSVP expression pattern during apical meristem determination suggested that its function is not to depress flower initiation but to maintain meristem development. Transcription of PtSVP in Arabidopsis svp-41 showed partially rescued SVP function. Ectopic overexpression of PtSVP in wild-type Arabidopsis induced late flowering similar to the phenotypes induced by other SVP/StMADS-11-like genes, but transformants produced additional trichomes and floral defects, such as flower-like structures instead of carpels. Ectopic expression of PtSVP in tobacco also caused additional florets. Overexpression of PtSVP in tobacco inhibited early transition of the coflorescence and prolonged coflorescence development, thus causing additional florets at the later stage. A yeast two-hybrid assay indicated that PtSVP significantly interacted with PtAP1, a homolog of Arabidopsis APETALA1 (AP1). These findings suggest that citrus SVP homolog genes are involved in flowering time regulation and may influence inflorescence meristem identity in some conditions or genetic backgrounds. SVP homologs might have evolved among plant species, but the protein functions are conserved between Arabidopsis and citrus.

Overexpression of a Kunitz-type trypsin inhibitor (AtKTI1) causes early flowering in Arabidopsis

An early flowering mutant of Arabidopsis, elf32-D was isolated from activation tagging screening. The mutant flowered earlier than wild type under both long day and short day conditions. The mutant phenotype was caused by overexpression of a Kunitz-type trypsin inhibitor gene (AtKTI1). The expression of AtKTI1 was detected in leaves, flowers, siliques and roots. In the vegetative state, no change of flowering integrator gene expression was observed for AtKTI1 overexpressing plants. In contrast, at the reproductive stage, its overexpression resulted in the down-regulation of FLC, a strong floral repressor which integrates the autonomous and vernalization pathways and also the up-regulation of FT and AP1, which are downstream floral integrator genes. It is probable that the AtKTI1 overexpression inhibits components of the flowering signaling pathway upstream of FLC, eventually regulating expression of FLC, or causing perturbations in plant metabolism and thus indirectly affecting flowering.

The significance of bolting and floral transitions as indicators of reproductive phase change in Arabidopsis

Reproductive phase change in Arabidopsis thaliana is characterized by the floral transition (initiation of the first flower) and the bolting transition (elongation of the first internode). Here, the relationship between these transitions is examined by comparing variation in cauline and total leaf numbers in wild-type plants and 49 early-flowering mutants under a wide range of photoperiods. The timing of these transitions was also evaluated by subjecting wild-type plants to photoperiodic perturbations at different developmental stages. Coupling between the bolting and floral transitions was altered in the wild type under non-optimal flowering conditions but could be restored by optimal conditions that activate the progression to flowering, including continuous light treatments and early flowering mutations. Under non-optimal photoperiodic conditions, the floral node was specified a few days before the bolting node. Altered definitions of long days for the cauline and total leaf responses were frequently coupled in early flowering mutants and were associated with similar photomorphogenetic defects. By contrast, altered definitions of short days were often opposite for the two leaf responses and were associated with different photomorphogenetic and circadian phenotypes. It is concluded that the bolting and floral transitions are regulated by different signalling pathways under non-optimal conditions and that phase change is a multidimensional process. This paper also proposes that, in contrast to the floral transition which is contingent on different factors, the bolting transition may be a robust indicator of reproductive phase change, especially when the progression to flowering is not optimal.

Highly efficient transformation of the GFP and MAC12.2 genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in Citrus

Precocious trifoliate orange (Poncirus trifoliata [L.] Raf), an extremely early flowering mutant of P. trifoliata, is an attractive model for functional genomics research in Citrus. A procedure for efficient regeneration and transformation of this genotype was developed by using green fluorescent protein (GFP) gene as visual marker and etiolated stem segments as explants. In vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic shoots and escapes. Transformation efficiency was 20.7% and the transformants were identified by polymerase chain reaction (PCR) and Southern blot analysis. Moreover, the transgenic lines expressed variable amounts of the GFP gene as revealed by real-time PCR analysis. Fifteen transgenic plants flowered 18 months after transfer to the greenhouse and six of them set fruits. GFP expression was also observed in the transgenic flowers and fruits. To test the utility of this system for functional genomics studies, an Arabidopsis thaliana MAC12.2 gene with the potential to produce seedless fruits was introduced into this genotype, and the traits of the transgenic fruits were characterized. The successful transformation of this perennial woody genotype with extremely short juvenility will allow us to test the function of cloned genes in citrus, the improvement of which is hindered by a long juvenility period.

Arabidopsis AMY1 expressions and early flowering mutant phenotype

The homozygous T-DNA mutant of the AMY1 gene in Arabidopsis was identified and importantly, shown to cause an early flowering phenotype. We found that the disruption of AMY1 enhanced expression of CO and FT. The expression analyses of genes related to starch metabolism revealed that expression of the AGPase small subunit APS1 in the wild type was higher than in the amy1 mutant. However, there were no significant differences in expression levels of the AGPase large subunit genes ApL1, AMY2, or AMY3 between wild type and the amy1 mutant. Expression profiling showed that AMY1 was highly expressed in leaves, stems, and flowers, and expressed less in leafstalks and roots. Furthermore, the level of AMY1 mRNA was highly elevated with age and in senescing leaves. RT-PCR analyses showed that the expression of AMY1 was induced by heat shock, GA, and ABA, while salt stress had no apparent effect on its expression.

Using precocious trifoliate orange ( Poncirus trifoliata [L.] Raf.) to establish a short juvenile transformation platform for citrus

The precocious trifoliate orange ( Poncirus trifoliata [L.] Raf.), an early flowering mutant of P. trifoliata, has a short juvenile phase of about 14 months, significantly shorter than other citrus. In this report, using the stems of precocious trifoliate orange seedlings as explants, an improved protocol mediated by Agrobacterium tumefaciens was developed to evaluate regeneration and transformation frequency. The transformed plants continued to reveal the precocious trait of non-transgenic mature precocious trifoliate orange, and as such could prove to be an efficient system for the verification of gene function in the fruits/flower via genetic transformation in woody plants.

Diversification of Photoperiodic Response Patterns in a Collection of Early-Flowering Mutants of Arabidopsis

Many plant species exhibit seasonal variation of flowering time in response to daylength. Arabidopsis (Arabidopsis thaliana) flowers earlier under long days (LDs) than under short days (SDs). This quantitative response to photoperiod is characterized by two parameters, the critical photoperiod (Pc), below which there is a delay in flowering, and the ceiling photoperiod (Pce), below which there is no further delay. Thus Pc and Pce define the thresholds beyond which maximum LD and SD responses are observed, respectively. We studied the quantitative response to photoperiod in 49 mutants selected for early flowering in SDs. Nine of these mutants exhibited normal Pce and Pc, showing that their precocious phenotype was not linked to abnormal measurement of daylength. However, we observed broad diversification in the patterns of quantitative responses in the other mutants. To identify factors involved in abnormal measurement of daylength, we analyzed the association of these various patterns with morphogenetic and rhythmic defects. A high proportion of mutants with altered Pce exhibited abnormal hypocotyl elongation in the dark and altered circadian periods of leaf movements. This suggested that the circadian clock and negative regulators of photomorphogenesis may contribute to the specification of SD responses. In contrast, altered Pc correlated with abnormal hypocotyl elongation in the light and reduced photosynthetic light-input requirements for bolting. This indicated that LD responses may be specified by positive elements of light signal transduction pathways and by regulators of resource allocation. Furthermore, the frequency of circadian defects in mutants with normal photoperiodic responses suggested that the circadian clock may regulate the number of leaves independently of its effect on daylength perception.

Identification of early-flower-related ESTs in an early-flowering mutant of trifoliate orange (Poncirus trifoliata) by suppression subtractive hybridization and macroarray analysis

To increase our understanding of the genes involved in flower development in citrus, we identified genes differentially expressed between juveniles and adults of an early-flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.) by suppressive subtractive hybridization (SSH) and macroarray hybridization analyses. Based on dot blot analysis, we confirmed that the juvenile subtracted cDNA library comprised genes upregulated during floral induction, inflorescence development and flowering. A total of 190 nonredundant expressed sequence tags were identified that were related flower development. The temporal and spatial expression patterns of four SSH-enriched genes were studied in adult precocious trifoliate orange by real-time PCR. Among these differentially expressed genes, the expression pattern of C5-B16 was closely correlated with inflorescence development and flowering. The full-length cDNA of C5-B16 was isolated by 5' and 3' rapid amplification of cDNA ends. Sequence analysis indicated that C5-B16 is a novel gene in citrus.

The FLX Gene of Arabidopsis is Required for FRI-Dependent Activation of FLC Expression

The Arabidopsis FLOWERING LOCUS C (FLC) gene encodes a MADS box protein that acts as a dose-dependent repressor of flowering. Mutants and ecotypes with elevated expression of FLC are late flowering and vernalization responsive. In this study we describe an early flowering mutant in the C24 ecotype, flc expressor (flx), that has reduced expression of FLC. FLX encodes a protein of unknown function with putative leucine zipper domains. FLX is required for FRIGIDA (FRI)-mediated activation of FLC but not for activation of FLC in autonomous pathway mutants. FLX is also required for expression of the FLC paralogs MADS AFFECTING FLOWERING 1 (MAF1) and MAF2.

Comparison of environmental and mutational variation in flowering time in Arabidopsis

Developmental dynamics can be influenced by external and endogenous factors in a more or less analogous manner. To compare the phenotypic effects of (i) environmental [i.e. standard (stPhP) and extended (exPhP) photoperiods] changes in Arabidopsis wild types and (ii) endogenous genetic variation in eav1-eav61 early flowering mutants, two temporal indicators were analysed, the time to bolting (DtB) and the number of leaves (TLN). It was found that DtB and TLN are differentially affected in different environmental and genetic contexts, and some factors of dynamic convergence were identified. The quantitative response to photoperiod is markedly contingent on the phototrophic input for DtB, but less so for TLN. To discriminate the light quantity and period components in DtB, two novel temporal indicators were determined, LtB (photosynthetic time to bolting) and PChron (DtB h(-1) of photoperiod), respectively. The use of PChron results in a coincidence of the variation profiles across stPhP and exPhP, interpreted as a buffering of the trophic response. Unlike natural accessions and later flowering mutants, the variation profiles across stPhP and eav mutants are significantly divergent, pointing to differences in environmental and genetic variation in flowering time. Yet, phenocopy effects and dynamic convergence between wild-type and mutant profiles are detected by using exPhP and the LtB indicator. Additional analyses of the cauline leaf number (CLN) show that the apical and basal boundaries of the primary inflorescence vary co-ordinately. The finding that the correlativity between CLN and TLN changes across photoperiods suggests that different states of intra-connectedness are involved in ontogenetic specification of flowering time and embodied in the primary inflorescence.

Arabidopsis Sucrose Transporter AtSUC9. High-Affinity Transport Activity, Intragenic Control of Expression, and Early Flowering Mutant Phenotype

AtSUC9 (At5g06170), a sucrose (Suc) transporter from Arabidopsis (Arabidopsis thaliana) L. Heynh., was expressed in Xenopus (Xenopus laevis) oocytes, and transport activity was analyzed. Compared to all other Suc transporters, AtSUC9 had an ultrahigh affinity for Suc (K(0.5) = 0.066 +/- 0.025 mm). AtSUC9 showed low substrate specificity, similar to AtSUC2 (At1g22710), and transported a wide range of glucosides, including helicin, salicin, arbutin, maltose, fraxin, esculin, turanose, and alpha-methyl-d-glucose. The ability of AtSUC9 to transport 10 glucosides was compared directly with that of AtSUC2, HvSUT1 (from barley [Hordeum vulgare]), and ShSUT1 (from sugarcane [Saccharum hybrid]), and results indicate that type I and type II Suc transporters have different substrate specificities. AtSUC9 protein was localized to the plasma membrane by transient expression in onion (Allium cepa) epidermis. Using a whole-gene translational fusion to beta-glucuronidase, AtSUC9 expression was found in sink tissues throughout the shoots and in flowers. AtSUC9 expression in Arabidopsis was dependent on intragenic sequence, and this was found to also be true for AtSUC1 (At1g71880) but not AtSUC2. Plants containing mutations in Suc transporter gene AtSUC9 were found to have an early flowering phenotype under short-day conditions. The transport properties of AtSUC9 indicate that it is uniquely suited to provide cellular uptake of Suc at very low extracellular Suc concentrations. The mutant phenotype of atsuc9 alleles indicates that AtSUC9 activity leads to a delay in floral transition.

SUPPRESSOR OF FRIGIDA3Encodes a Nuclear ACTIN-RELATED PROTEIN6 Required for Floral Repression inArabidopsis w⃞

Flowering traits in winter annual Arabidopsis thaliana are conferred mainly by two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). FLC acts as a flowering repressor and is regulated by multiple flowering pathways. We isolated an early-flowering mutant, suppressor of FRIGIDA3 (suf3), which also shows leaf serration, weak apical dominance, and infrequent conversion of the inflorescence shoot to a terminal flower. The suf3 mutation caused a decrease in the transcript level of FLC in both a FRI-containing line and autonomous pathway mutants. However, suf3 showed only a partial reduction of FLC transcript level, although it largely suppressed the late-flowering phenotype. In addition, the suf3 mutation caused acceleration of flowering in both 35S-FLC and a flc null mutant, indicating that SUF3 regulates additional factor(s) for the repression of flowering. SUF3 is highly expressed in the shoot apex, but the expression is not regulated by FRI, autonomous pathway genes, or vernalization. SUF3 encodes the nuclear ACTIN-RELATED PROTEIN6 (ARP6), the homolog of which in yeast is a component of an ATP-dependent chromatin-remodeling SWR1 complex. Our analyses showed that SUF3 regulates FLC expression independent of vernalization, FRI, and an autonomous pathway gene, all of which affect the histone modification of FLC chromatin. Subcellular localization using a green fluorescent protein fusion showed that Arabidopsis ARP6 is located at distinct regions of the nuclear periphery.

Genetic mapping of Eef1, a major effect QTL for early flowering in Eucalyptus grandis

An early flowering mutant plant of Eucalyptus grandis with normal vegetative growth was found in a nursery in northern Brazil. This mutant plant flowers at approximately 90 days from germination. A cross between a wild-type (normal flowering) tree and the mutant was carried out, generating a progeny of 88 individuals where early flowering segregated in an approximate 1:1 ratio. A genome scan with 100 microsatellite markers distributed across the genome was carried out using bulk segregant analysis (BSA) on two contrasting bulks of 15 plants each. Linkages (LOD>3.0) with a major effect early flowering quantitative trait locus (QTL) were detected and confirmed by a full scale cosegregation analysis for markers EMBRA27, EMBRA60, EMBRA164, EMBRA158, EMBRA91, and EMBRA65. A localized linkage map involving the six loci and the early flowering QTL named Eucalyptus early flowering 1 (Eef1) was constructed belonging to linkage group #2 in the existing microsatellite reference map. The Eef1 locus was mapped between markers EMBRA27 and EMBRA164, with distances of 21.8 and 6.4 cM, respectively. In introgression experiments, these two markers could be successfully used with an expected precision of 98% to select plants carrying the Eef1 mutant allele, assuming no recombination interference in the genomic segment. Early flowering could be a very useful trait both in breeding as well as experimental genetics of Eucalyptus.

Registration of LGRU ef Early Flowering Mutant of Rice

Registration of LGRU <i>ef</i> Early Flowering Mutant of Rice

Extensive phenotypic variation in early flowering mutants of Arabidopsis.

Flowering time, the major regulatory transition of plant sequential development, is modulated by multiple endogenous and environmental factors. By phenotypic profiling of 80 early flowering mutants of Arabidopsis, we examine how mutational reduction of floral repression is associated with changes in phenotypic plasticity and stability. Flowering time measurements in mutants reveal deviations from the linear relationship between the number of leaves and number of days to bolting described for natural accessions and late flowering mutants. The deviations correspond to relative early bolting and relative late bolting phenotypes. Only a minority of mutants presents no detectable phenotypic variation. Mutants are characterized by a broad release of morphological pleiotropy under short days, with leaf characters being most variable. They also exhibit changes in phenotypic plasticity across environments for florigenic-related responses, including the reaction to light and dark, photoperiodic behavior, and Suc sensitivity. Morphological pleiotropy and plasticity modifications are differentially distributed among mutants, resulting in a large diversity of multiple phenotypic changes. The pleiotropic effects observed may indicate that floral repression defects are linked to global developmental perturbations. This first, to our knowledge, extensive characterization of phenotypic variation in early flowering mutants correlates with the reports that most factors recruited in floral repression at the molecular genetic level correspond to ubiquitous regulators. We discuss the importance of functional ubiquity for floral repression with respect to robustness and flexibility of network biological systems.