The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis

The flowering time of plants is affected by modest changes in ambient temperature. However, little is known about the regulation of ambient temperature-responsive flowering by small RNAs. In this study, we show that the microRNA156 (miR156)-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) module directly regulates FLOWERING LOCUS T (FT) expression in the leaf to control ambient temperature-responsive flowering. Overexpression of miR156 led to more delayed flowering at a lower ambient temperature (16°C), which was associated with down-regulation of FT and FRUITFULL expression. Among miR156 target genes, SPL3 mRNA levels were mainly reduced, probably because miR156-mediated cleavage of SPL3 mRNA was higher at 16°C. Overexpression of miR156-resistant SPL3 [SPL3(-)] caused early flowering, regardless of the ambient temperature, which was associated with up-regulation of FT and FRUITFULL expression. Reduction of miR156 activity by target mimicry led to a phenotype similar to that of SUC2::rSPL3 plants. FT up-regulation was observed after dexamethasone treatment in GVG-rSPL3 plants. Misexpression and artificial microRNA-mediated suppression of FT in the leaf dramatically altered the ambient temperature-responsive flowering of plants overexpressing miR156 and SPL3(-). Chromatin immunoprecipitation assay showed that the SPL3 protein directly binds to GTAC motifs within the FT promoter. Lesions in TERMINAL FLOWER1, SHORT VEGETATIVE PHASE, and EARLY FLOWERING3 did not alter the expression of miR156 and SPL3. Taken together, our data suggest that the interaction between the miR156-SPL3 module and FT is part of the regulatory mechanism controlling flowering time in response to ambient temperature.     

Genetic networks regulated by ASYMMETRIC LEAVES1 (AS1) and AS2 in leaf development in Arabidopsis thaliana: KNOX genes control five morphological events

The asymmetric leaves 1 ( as1 ) and as2 mutants of Arabidopsis thaliana exhibit pleiotropic phenotypes. Expression of a number of genes, including three class-1 KNOTTED -like homeobox ( KNOX ) genes ( BP , KNAT2 and KNAT6 ) and ETTIN / ARF3 , is enhanced in these mutants. In the present study, we attempted to identify the phenotypic features of as1 and as2 mutants that were generated by ectopic expression of KNOX genes, using multiple loss-of-function mutations of KNOX genes as well as as1 and as2 . Our results revealed that the ectopic expression of class-1 KNOX genes resulted in reductions in the sizes of leaves, reductions in the size of sepals and petals, the formation of a less prominent midvein, the repression of adventitious root formation and late flowering. Our results also revealed that the reduction in leaf size and late flowering were caused by the repression, by KNOX genes, of a gibberellin (GA) pathway in as1 and as2 plants. The formation of a less prominent midvein and the repression of adventitious root formation were not, however, related to the GA pathway. The asymmetric formation of leaf lobes, the lower complexity of higher-ordered veins, and the elevated frequency of adventitious shoot formation on leaves of as1 and as2 plants were not rescued by multiple mutations in KNOX genes. These features must, therefore, be controlled by other genes in which expression is enhanced in the as1 and as2 mutants.     

The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity

The ABC model of flower organ identity is widely recognized as providing a framework for understanding the specification of flower organs in diverse plant species. Recent studies in Arabidopsis thaliana have shown that three closely related MADS-box genes, SEPALLATA1 (SEP1), SEP2 and SEP3, are required to specify petals, stamens, and carpels because these organs are converted into sepals in sep1 sep2 sep3 triple mutants. Additional studies indicate that the SEP proteins form multimeric complexes with the products of the B and C organ identity genes. Here, we characterize the SEP4 gene, which shares extensive sequence similarity to and an overlapping expression pattern with the other SEP genes. Although sep4 single mutants display a phenotype similar to that of wild-type plants, we find that floral organs are converted into leaf-like organs in sep1 sep2 sep3 sep4 quadruple mutants, indicating the involvement of all four SEP genes in the development of sepals. We also find that SEP4 contributes to the development of petals, stamens, and carpels in addition to sepals and that it plays an important role in meristem identity. These and other data demonstrate that the SEP genes play central roles in flower meristem identity and organ identity.     

Allele-specific effects of PDF2 on floral morphology in Arabidopsis thaliana

The class IV Homeodomain-leucine zipper (HD-ZIP IV) gene family includes several genes that are functionally significant in epidermal development. Our recent study revealed that double mutants of the epidermis-expressed HD-ZIP IV members, PROTODERMAL FACTOR2 (PDF2) in combination with some HOMEODOMAIN GLABROUS (HDG, pronounced “hedge”) genes, affect stamen development and specification of petal and stamen identity, possibly in a non cell-autonomous manner. However, the effect of the pdf2 mutations on the floral development was largely different depending on T-DNA insertion locations: pdf2–1 hdg flowers exhibited homeotic conversion of petals and stamens, while pdf2–2 hdg flowers had only a reduced number of stamens. Here, we used 2 additional pdf2 alleles to make double mutants and found that their floral phenotypes were rather similar to those of pdf2–2 hdg. The allele-specific effect caused by pdf2–1, which carries a T-DNA in a steroidogenic acute regulatory protein-related lipid transfer (START) domain-encoding region, suggests the importance of the START domain in proper function of HD-ZIP IV proteins.     

Correlative controls of senescence and plant death in Arabidopsis thaliana (Brassicaceae)

Like most monocarpic plants, longevity of Arabidopsis thaliana plants is controlled by the reproductive structures; however, they appear to work differently from most dicots studied. Neither male- and female-sterility mutations (ms1-1 and bell1, respectively) nor surgical removal of the stems with inflorescences (bolts) at various stages significantly increased the longevity of individual rosette leaves, yet the mutants and treated plants lived 20-50 d longer, measured by the death of the last rosette and/or the last cauline leaf. A series of growth mutations (clv2-4, clv3-2, det3, vam1 enh, and dark green) also increased plant longevity by 20-30 d but did not delay the overall development of the plants. The mutations prolonged plant life through the production of new leaves and stems with inflorescences (bolts) rather than by extending leaf longevity. In growing stems, the newly-formed leaves may induce senescence in the older leaves; however, removal of the younger leaves did not significantly increase the life of the older leaves on the compressed stems of Arabidopsis. Since plants that produce more bolts also live longer, the reproductive load (dry weight) of the bolts did not seem to drive leaf or whole plant senescence here. The developing reproductive structures caused the death of the plant by preventing regeneration of leaves and bolts, which are green and presumably photosynthetic. They also exerted a correlative control (repression) on the development of additional reproductive structures.     

Mutual regulation of Arabidopsis thaliana ethylene-responsive element binding protein and a plant floral homeotic gene, APETALA2

BACKGROUND AND AIMS: It has previously been shown that Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP) contributed to resistance to abiotic stresses. Interestingly, it has also been reported that expression of ethylene-responsive factor (ERF) genes including AtEBP were regulated by the activity of APETALA2 (AP2), a floral homeotic factor. AP2 is known to regulate expression of several floral-specific homeotic genes such as AGAMOUS. The aim of this study was to clarify the relationship between AP2 and AtEBP in gene expression. METHODS: Northern blot analysis was performed on ap2 mutants, ethylene-related Arabidopsis mutants and transgenic Arabidopsis plants over-expressing AtEBP, and a T-DNA insertional mutant of AtEBP. Phenotypic analysis of these plants was performed. KEY RESULTS: Expression levels of ERF genes such as AtEBP and AtERF1 were increased in ap2 mutants. Over-expression of AtEBP caused upregulation of AP2 expression in leaves. AP2 expression was suppressed by the null-function of ethylene-insensitive2 (EIN2), although AP2 expression was not affected by ethylene treatment. Loss of AtEBP function slightly reduced the average number of stamens. CONCLUSIONS: AP2 and AtEBP are mutually regulated in terms of gene expression. AP2 expression was affected by EIN2 but was not regulated by ethylene treatment.     

Histone deacetylases and ASYMMETRIC LEAVES2 are involved in the establishment of polarity in leaves of Arabidopsis

We show that two Arabidopsis thaliana genes for histone deacetylases (HDACs), HDT1/HD2A and HDT2/HD2B, are required to establish leaf polarity in the presence of mutant ASYMMETRIC LEAVES2 (AS2) or AS1. Treatment of as1 or as2 plants with inhibitors of HDACs resulted in abaxialized filamentous leaves and aberrant distribution of microRNA165 and/or microRNA166 (miR165/166) in leaves. Knockdown mutations of these two HDACs by RNA interference resulted in phenotypes like those observed in the as2 background. Nuclear localization of overproduced AS2 resulted in decreased levels of mature miR165/166 in leaves. This abnormality was abolished by HDAC inhibitors, suggesting that HDACs are required for AS2 action. A loss-of-function mutation in HASTY, encoding a positive regulator of miRNA levels, and a gain-of-function mutation in PHABULOSA, encoding a determinant of adaxialization, suppressed the generation of abaxialized filamentous leaves by inhibition of HDACs in the as1 or as2 background. AS2 and AS1 were colocalized in subnuclear bodies adjacent to the nucleolus where HDT1/HD2A and HDT2/HD2B were also found. Our results suggest that these HDACs and both AS2 and AS1 act independently to control levels and/or patterns of miR165/166 distribution and the development of adaxial-abaxial leaf polarity and that there may be interactions between HDACs and AS2 (AS1) in the generation of those miRNAs.     

HUA2 is required for the expression of floral repressors in Arabidopsis thaliana

The HUA2 gene acts as a repressor of floral transition. Lesions in hua2 were identified through a study of natural variation and through two mutant screens. An allele of HUA2 from Landsberg erecta (Ler) contains a premature stop codon and acts as an enhancer of early flowering 4 (elf4) mutants. hua2 single mutants, in the absence of the elf4 lesion, flower earlier than wild type under short days. hua2 mutations partially suppress late flowering in FRIGIDA (FRI )-containing lines, autonomous pathway mutants, and a photoperiod pathway mutant. hua2 mutations suppress late flowering by reducing the expression of several MADS genes that act as floral repressors including FLOWERING LOCUS C (FLC ) and FLOWERING LOCUS M (FLM ).