AGO1 controls arabidopsis inflorescence architecture possibly by regulating TFL1 expression

Background and Aims

The TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators.

Methods

Mutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (β-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype.

Key Results

A mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants.

Conclusions

The results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression.     

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.     

Virus-induced gene silencing of PEAM4 affects floral morphology by altering the expression pattern of PsSOC1a and PsPVP in pea

pea-MADS4 (PEAM4) regulates floral morphology in Pisum sativum L., however, its molecular mechanisms still remain unclear. Virus-induced gene silencing (VIGS) is a recently developed reverse genetic approach that facilities an easier and more rapid study of gene functions. In this study, the PEAM4 gene was effectively silenced by VIGS using a pea early browning virus (PEBV) in wild type pea JI992. The infected plants showed abnormal phenotypes, as the floral organs, especially the sepals and petals changed in both size and shape, which made the corolla less closed. The petals changed in morphology and internal symmetry with, the stamens reduced and carpel dehisced. Larger sepals and longer tendrils with small cauline leaves appeared, with some sepals turning into bracts, and secondary inflorescences with fused floral organs were formed, indicating a flower-to-inflorescence change. The infected plants also displayed a delayed and prolonged flowering time. The PEAM4-VIGS plants with altered floral morphology were similar to the pim (proliferating inflorescence meristem) mutant and also mimicked the phenotypes of ap1 mutants in Arabidopsis. The expression pattern of the homologous genes PsSOC1a and PsSVP, which were involved in flowering time and florescence morphological control downstream of PEAM4, were analyzed by real-time RT-PCR and mRNA in situ hybridization. PsSOC1a and PsSVP were ectopically expressed and enhanced in the floral meristems from PEAM4-silenced plants. Our data suggests that PEAM4 may have a similar molecular mechanism as AtAP1, which inhibits the expression of PsSOC1a and PsSVP in the floral meristem from the early stages of flower development. As such, in this way PEAM4 plays a crucial role in maintaining floral organ identity and flower development in pea.     

UFO in the Arabidopsis inflorescence apex is required for floral-meristem identity and bract suppression

The UNUSUAL FLORAL ORGANS (UFO) gene of Arabidopsis encodes an F-box protein required for the determination of floral-organ and floral-meristem identity. Mutation of UFO leads to dramatic changes in floral-organ type which are well-characterized whereas inflorescence defects are more subtle and less understood. These defects include an increase in the number of secondary inflorescences, nodes that alternate between forming flowers and secondary inflorescences, and nodes in which a single flower is subtended by a bract. Here, we show how inflorescence defects correlate with the abnormal development of floral primordia and establish a temporal requirement for UFO in this process. At the inflorescence apex of ufo mutants, newly formed primordia are initially bract-like. Expression of the floral-meristem identity genes LFY and AP1 are confined to a relatively small adaxial region of these primordia with expression of the bract-identity marker FIL observed in cells that comprise the balance of the primordia. Proliferation of cells in the adaxial region of these early primordia is delayed by several nodes such that primordia appear “chimeric” at several nodes, having visible floral and bract components. However, by late stage 2 of floral development, growth of the bract generally ceases and is overtaken by development of the floral primordium. This abnormal pattern of floral meristem development is not rescued by expression of UFO from the AP1 promoter, indicating that UFO is required prior to AP1 activation for normal development of floral primordia. We propose that UFO and LFY are jointly required in the inflorescence meristem to both promote floral meristem development and inhibit, in a non-cell autonomous manner, growth of the bract.Shelley R. Hepworth and Jennifer E. Klenz contributed equally to this work.     

Hormone-regulated inflorescence induction and TFL1 expression in Arabidopsis callus in vitro

To study hormone-regulated inflorescence development, we established the in vitro regeneration system of Arabidopsis inflorescences in the presence of cytokinin and auxin. Media containing a combination of thidiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) were used to induce callus formation. Higher frequencies of calli were obtained by using the inflorescence stems as explants. After transferring the calli to media containing a combination of zeatin and indole-3-acetic acid (IAA), the inflorescences were induced from the calli. The morphology of regenerated inflorescences was similar to that of inflorescences in plants; however, flowers of regenerated inflorescences often lacked a few floral organs. Furthermore, TFL1, a gene involved in floral transition in Arabidopsis, was activated during the inflorescence induction. Our results suggest that the TFL1 gene plays an important role in hormone-regulated inflorescence formation.     

BROTHER OF FT AND TFL1 (BFT) has TFL1‐like activity and functions redundantly with TFL1 in inflorescence meristem development in Arabidopsis

The FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family is a small gene family that encodes important regulators that control flower development in Arabidopsis. Here, we investigated the biological role of the product of BROTHER OF FT AND TFL1 (BFT), a member of this family, whose function remains unknown. Comparison of the critical residues that play a role in distinguishing FT‐ or TFL1‐like activity revealed that BFT is more similar to FT. Similar to FT expression, BFT expression showed a diurnal oscillation pattern, peaking in the evening. In situ hybridization revealed BFT expression in the shoot apical meristem, young leaf and axillary inflorescence meristem. Transgenic plants over‐expressing BFT exhibited delayed flowering and severe floral defects (floral indeterminacy and compact inflorescences surrounded by serrate leaves), similar to 35S::TFL1 plants. LEAFY (LFY) and APETALA1 (AP1) expression was significantly reduced in 35S::BFT plants. BFT over‐expression failed to rescue the terminal flower phenotype of tfl1 mutants; however, it delayed both terminal flower formation in the primary inflorescence and axillary inflorescence development in the tfl1 mutant background. Consistent with this, the loss‐of‐function BFT alleles, bft‐2 and an BFT RNAi line, accelerated termination of the primary inflorescence and formation of axillary inflorescences in the tfl1 mutant background. Taken together, our results suggest that, despite similarities in the critical residues of BFT and FT, BFT possesses a TFL1‐like activity and functions redundantly with TFL1 in inflorescence meristem development, and possibly contributes to the regulation of plant architecture.     

Characterization of waldmeister, a novel developmental mutant in Arabidopsis thaliana

A novel Arabidopsis thaliana (L.) Heynh. developmental mutant,waldmeister (wam), is described. This mutant was found in theprogeny arising from an Ac-Ds tagging experiment, but does notappear to be tagged by an introduced transposon. This recessivenuclear mutation maps between GAPB and ap1 on chromosome 1 andshows extreme morphological and physiological changes in bothfloral and vegetative tissues. Changes to the vegetative phenotypeinclude altered leaf morphology, multiple rosettes, stem fasciation,retarded senescence and disturbed geotropic growth. Changesto the floral phenotype include delayed flowering, increasednumber of inflorescences, determinate inflorescences, alterednumber and morphology of floral organs, chimeric floral organs,and ectopic ovules . wam was crossed to a number of previouslydescribed floral mutants: apetela 2, apetela 3, pistillata,agamous, and leafy. The phenotype of the double mutant was ineach case additive. In the case of agamous, however, the indeterminaterepetitive floral structure of agamous was lacking, emphasizingthe determinate inflorescence growth of wam. The extreme phenotypeof the wam mutant is suggestive of a disturbance to a gene ofglobal importance in the regulation of plant growth and development. Key words: Arabidopsis thaliana, waldmeister, developmental mutant, flower mutant     

Rate heterogeneity in six protein-coding genes from the holoparasite Balanophora (Balanophoraceae) and other taxa of Santalales

Background and Aims

The holoparasitic flowering plant Balanophora displays extreme floral reduction and was previously found to have enormous rate acceleration in the nuclear 18S rDNA region. So far, it remains unclear whether non-ribosomal, protein-coding genes of Balanophora also evolve in an accelerated fashion and whether the genes with high substitution rates retain their functionality. To tackle these issues, six different genes were sequenced from two Balanophora species and their rate variation and expression patterns were examined.

Methods

Sequences including nuclear PI, euAP3, TM6, LFY and RPB2 and mitochondrial matR were determined from two Balanophora spp. and compared with selected hemiparasitic species of Santalales and autotrophic core eudicots. Gene expression was detected for the six protein-coding genes and the expression patterns of the three B-class genes (PI, AP3 and TM6) were further examined across different organs of B. laxiflora using RT-PCR.

Key Results

Balanophora mitochondrial matR is highly accelerated in both nonsynonymous (d_N) and synonymous (d_S) substitution rates, whereas the rate variation of nuclear genes LFY, PI, euAP3, TM6 and RPB2 are less dramatic. Significant d_S increases were detected in Balanophora PI, TM6, RPB2 and d_N accelerations in euAP3. All of the protein-coding genes are expressed in inflorescences, indicative of their functionality. PI is restrictively expressed in tepals, synandria and floral bracts, whereas AP3 and TM6 are widely expressed in both male and female inflorescences.

Conclusions

Despite the observation that rates of sequence evolution are generally higher in Balanophora than in hemiparasitic species of Santalales and autotrophic core eudicots, the five nuclear protein-coding genes are functional and are evolving at a much slower rate than 18S rDNA. The mechanism or mechanisms responsible for rapid sequence evolution and concomitant rate acceleration for 18S rDNA and matR are currently not well understood and require further study in Balanophora and other holoparasites.     

Ectopic expression of FaesAP3, a Fagopyrum esculentum (Polygonaceae) AP3 orthologous gene rescues stamen development in an Arabidopsis ap3 mutant

Arabidopsis thaliana APETALA3 (AP3) and Antirrhinum majus DEFICIENS (DEF) MADS box genes are required to specify petal and stamen identity. AP3 and DEF are members of the euAP3 lineage, which arose by gene duplication coincident with radiation of the core eudicots. In order to investigate the molecular mechanisms underlying organ development in early diverging clades of core eudicots, we isolated and identified an AP3 homolog, FaesAP3, from Fagopyrum esculentum (buckwheat, Polygonaceae), a multi-food-use pseudocereal with healing benefits. Protein sequence alignment and phylogenetic analyses revealed that FaesAP3 grouped into the euAP3 lineage. Expression analysis showed that FaesAP3 was transcribed only in developing stamens, and differed from AP3 and DEF, which expressed in developing petals and stamens. Moreover, ectopic expression of FaesAP3 rescued stamen development without complementation of petal development in an Arabidopsis ap3 mutant. Our results suggest that FaesAP3 is involved in the development of stamens in buckwheat. These results also suggest that FaesAP3 holds some potential for biotechnical engineering to create a male sterile line of F. esculentum.     

Pleiotropic and nonredundant effects of an auxin importer in Setaria and maize

Directional transport of auxin is critical for inflorescence and floral development in flowering plants, but the role of auxin influx carriers (AUX1 proteins) has been largely overlooked. Taking advantage of available AUX1 mutants in green millet (Setaria viridis) and maize (Zea mays), we uncover previously unreported aspects of plant development that are affected by auxin influx, including higher order branches in the inflorescence, stigma branch number, glume (floral bract) development, and plant fertility. However, disruption of auxin flux does not affect all parts of the plant, with little obvious effect on inflorescence meristem size, time to flowering, and anther morphology. In double mutant studies in maize, disruptions of ZmAUX1 also affect vegetative development. A green fluorescent protein (GFP)-tagged construct of the Setaria AUX1 protein Sparse Panicle1 (SPP1) under its native promoter showed that SPP1 localizes to the plasma membrane of outer tissue layers in both roots and inflorescences, and accumulates specifically in inflorescence branch meristems, consistent with the mutant phenotype and expected auxin maxima. RNA-seq analysis indicated that most gene expression modules are conserved between mutant and wild-type plants, with only a few hundred genes differentially expressed in spp1 inflorescences. Using clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 technology, we disrupted SPP1 and the other four AUX1 homologs in S. viridis. SPP1 has a larger effect on inflorescence development than the others, although all contribute to plant height, tiller formation, and leaf and root development. The AUX1 importers are thus not fully redundant in S. viridis. Our detailed phenotypic characterization plus a stable GFP-tagged line offer tools for future dissection of the function of auxin influx proteins.

Mutations in a single auxin importer gene uncover broad and unexpected effects in nearly all aspects of the development of shoots, inflorescences, and flowers.     

Fatty acid desaturase-6 (Fad6) is required for salt tolerance in Arabidopsis thaliana

Fatty acid desaturases play important role in plant responses to abiotic stresses including cold, high temperature, drought, and osmotic stress. In this work, we provide the evidence that Fad6, a chloroplast desaturase, is required for salt tolerance during the early seedling development of Arabidopsis. Expression of Fad6 was responsive to salt and osmotic stress. Compared with the wild-type plants, the fad6 mutant showed reduced tolerance to salt stress, and accumulated more Na⁺ and less K⁺ under high NaCl stress condition. Furthermore, cellular oxidative damage was more severe in fad6 when treated with high concentrations of NaCl, as indicated by increased electrolyte leakage rate and malondialdehyde production, as well as by decreased activities of anti-oxidative enzymes. All these results suggest that Fad6 is required for salt resistance in Arabidopsis.