Arabidopsis thaliana CENTRORADIALIS homologue (ATC) acts systemically to inhibit floral initiation in Arabidopsis

Floral initiation is orchestrated by systemic floral activators and inhibitors. This remote‐control system may integrate environmental cues to modulate floral initiation. Recently, FLOWERING LOCUS T (FT) was found to be a florigen. However, the identity of systemic floral inhibitor or anti‐florigen remains to be elucidated. Here we show that Arabidopsis thaliana CENTRORADIALIS homologue (ATC), an Arabidopsis FT homologue, may act in a non‐cell autonomous manner to inhibit floral initiation. Analysis of the ATC null mutant revealed that ATC is a short‐day‐induced floral inhibitor. Cell type‐specific expression showed that companion cells and apex that express ATC are sufficient to inhibit floral initiation. Histochemical analysis showed that the promoter activity of ATC was mainly found in vasculature but under the detection limit in apex, a finding that suggests that ATC may move from the vasculature to the apex to influence flowering. Consistent with this notion, Arabidopsis seedling grafting experiments demonstrated that ATC moved over a long distance and that floral inhibition by ATC is graft transmissible. ATC probably antagonizes FT activity, because both ATC and FT interact with FD and affect the same downstream meristem identity genes APETALA1, in an opposite manner. Thus, photoperiodic variations may trigger functionally opposite FT homologues to systemically influence floral initiation.     

AXL and AXR1 have redundant functions in RUB conjugation and growth and development in Arabidopsis

Cullin-RING ubiquitin-protein ligases such as the Skp1, cullin, F-box protein (SCF) have been implicated in many growth and developmental processes in plants. Normal SCF function requires that the CUL1 subunit be post-translationally modified by related to ubiquitin (RUB), a protein related to ubiquitin. This process is mediated by two enzymes: the RUB-activating and RUB-conjugating enzymes. In Arabidopsis, the RUB-activating enzyme is a heterodimer consisting of AXR1 and ECR1. Mutations in the AXR1 gene result in a pleiotropic phenotype that includes resistance to the plant hormone auxin. Here we report that the AXL (AXR1-like) gene also functions in the RUB conjugation pathway. Overexpression of AXL in the axr1-3 background complements the axr1-3 phenotype. Biochemical analysis indicates that AXL overexpression restores CUL1 modification to the wild-type level, indicating that AXR1 and AXL have the same biochemical activity. Although the axl mutant resembles wild-type plants, the majority of axr1 axl-1 double mutants are embryo or seedling lethal. Furthermore, the axl-1 mutation reveals novel RUB-dependent processes in embryo development. We conclude that AXR1 and AXL function redundantly in the RUB conjugating pathway.     

NAP1 acts with Clb1 to perform mitotic functions and to suppress polar bud growth in budding yeast

NAP1 is a 60-kD protein that interacts specifically with mitotic cyclins in budding yeast and frogs. We have examined the ability of the yeast mitotic cyclin Clb2 to function in cells that lack NAP1. Our results demonstrate that Clb2 is unable to carry out its full range of functions without NAP1, even though Clb2/p34CDC28-associated kinase activity rises to normal levels. In the absence of NAP1, Clb2 is unable to efficiently induce mitotic events, and cells undergo a prolonged delay at the short spindle stage with normal levels of Clb2/p34CDC28 kinase activity. NAP1 is also required for the ability of Clb2 to induce the switch from polar to isotropic bud growth. As a result, polar bud growth continues during mitosis, giving rise to highly elongated cells. Our experiments also suggest that NAP1 is required for the ability of the Clb2/p34CDC28 kinase complex to amplify its own production, and that NAP1 plays a role in regulation of microtubule dynamics during mitosis. Together, these results demonstrate that NAP1 is required for the normal function of the activated Clb2/p34CDC28 kinase complex, and provide a step towards understanding how cyclin- dependent kinase complexes induce specific events during the cell cycle.     

Tumour-promoting phorbol esters rapidly inhibit bud formation in hydra

Summary The effects of tumour promoters and carcinogens on bud formation were investigated in an attempt to clarify the primary process of bud formation in hydra. Treatment with 1.0ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-didecanoate (PDD) or mezerein added immediately after feeding rapidly and completely inhibited the formation of new buds in Hydra japonica. Treatment with TPA 3–6 h after feeding also suppressed bud formation 24 h later, but suppressed buds appeared 48 h later. Buds suppressed by TPA also formed in the presence of a diluted homogenate of hydra and during starvation. Carcinogens, such as benzo(a)pyrene and 20-methylcholanthrene, did not have an inhibitory effect on bud formation within 2 days. The tumour promoters and carcinogens used in this experiment did not inhibit the regeneration of tentacles. These results indicate that tumour-promoting phorbol esters, but not carcinogens, rapidly suppress the process by which the formation of buds is initiated by hydra, and the effects of these esters depend on the timing of treatment after feeding.     

AXR1 is involved in BR-mediated elongation and SAUR-AC1 gene expression in Arabidopsis

Limited information is available concerning the interactions between the brassinosteroid (BR) and auxin signaling pathways. The expression pattern of the SAUR-AC1 gene, an early auxin-inducible gene in Arabidopsis, was studied in response to brassinolide (BL), in the presence of a BR-biosynthesis inhibitor, in a BR-deficient mutant, and in combination with auxin. The results suggested that the SAUR-AC1 gene is regulated by BRs independently of auxin levels, and that it is important in BR-mediated elongation. The axr1 (auxin insensitive 1) mutant was less sensitive to BL-induced elongation and BL-induced SAUR-AC1 expression, suggesting that a ubiquitin ligase-mediated system is involved in BR-mediated elongation.     

MM31/EIR1 promotes lateral root formation in Arabidopsis

Lateral root formation in Arabidopsis provides a model for the study of auxin function. Tryptophan (Trp) is a precursor of the auxin indoleacetic acid (IAA). To study the physiological function of Trp in auxin-related phenotypes, we examined the effect of Trp on lateral root formation. We found that Trp treatment enhanced lateral root formation and, by screening for mutants in which the effect of Trp on lateral root formation was enhanced, we isolated the mm31 mutant. Based on genetic and physiological analyses, we propose that MM31/EIR1 modulates lateral root formation by regulating the IAA polar transport system, and that auxin transport from the shoot to the root regulates lateral root formation.Key words: lateral root formation, Arabidopsis, EIR1, IAA, auxin     

The AXR1 and AUX1 genes of Arabidopsis function in separate auxin-response pathways

The recessive mutations aux1 and axr1 of Arabidopsis confer resistance to the plant hormone auxin. The axr1 mutants display a variety of morphological defects. In contrast, the only morphological defect observed in aux1 mutants is a loss of root gravitropism. To learn more about the function of these genes in auxin response, the expression of the auxin-regulated gene SAUR-AC1 in mutant and wild-type plants has been examined. It has been found that axr1 plants display a pronounced deficiency in auxin-induced accumulation of SAUR-AC1 mRNA in seedlings as well as rosette leaves and mature roots. In contrast, the aux1 mutation has a modest effect on auxin induction of SAUR-AC1. To determine if the AUX1 and AXR1 genes interact to facilitate auxin response, plants which are homozygous for both aux1 and axr1 mutations have been constructed and characterized. The two mutations are additive in their effects on auxin response, suggesting that each mutation confers resistance by a different mechanism. However, the morphology of double mutant plants indicates that there is an inter-action between the AXR1 and AUX1 genes. In mature plants, the aux1-7 mutation acts to partially suppress the morphological defects conferred by the axr1-12 mutation. This suppression is not accompanied by an increase in auxin response, as measured by SAUR-AC1 expression, suggesting that the interaction between the AUX1 and AXR1 genes is indirect.     

The hormonal regulation of axillary bud growth in Arabidopsis

Apically derived auxin has long been known to inhibit lateral bud growth, but since it appears not to enter the bud, it has been proposed that its inhibitory effect is mediated by a second messenger. Candidates include the plant hormones ethylene, cytokinin and abscisic acid. We have developed a new assay to study this phenomenon using the model plant Arabidopsis. The assay allows study of the effects of both apical and basal hormone applications on the growth of buds on excised nodal sections. We have shown that apical auxin can inhibit the growth of small buds, but larger buds were found to have lost competence to respond. We have used the assay with nodes from wild-type and hormone-signalling mutants to test the role of ethylene, cytokinin and abscisic acid in bud inhibition by apical auxin. Our data eliminate ethylene as a second messenger for auxin-mediated bud inhibition. Similarly, abscisic acid signalling is not to be required for auxin action, although basally applied abscisic can enhance inhibition by apical auxin and apically applied abscisic acid can reduce it. By contrast, basally applied cytokinin was found to release lateral buds from inhibition by apical auxin, while apically applied cytokinin dramatically increased the duration of inhibition. These results are consistent with cytokinin acting independently to regulate bud growth, rather than as a second messenger for auxin. However, in the absence of cytokinin-signalling mutants, a role for cytokinin as a second messenger for auxin cannot be ruled out.     

AXR1 promotes the Arabidopsis cytokinin response by facilitating ARR5 proteolysis

The plant hormone cytokinin plays essential roles in many aspects of growth and development. The cytokinin signal is transmitted by a multi‐step phosphorelay to the members of two functionally antagonistic classes of Arabidopsis response regulators (ARRs): type B ARRs (response activators) and type A ARRs (negative‐feedback regulators). Previous studies have shown that mutations in AXR1, encoding a subunit of the E1 enzyme in the RUB (related to ubiquitin) modification pathway, lead to decreased cytokinin sensitivity. Here we show that the cytokinin resistance of axr1 seedlings is suppressed by loss of function of the type A ARR family member ARR5. Based on the established role of the RUB pathway in ubiquitin‐dependent proteolysis, these data suggest that AXR1 promotes the cytokinin response by facilitating type A ARR degradation. Indeed, both genetic (axr1 mutants) and chemical (MLN4924) suppression of RUB E1 increased ARR5 stability, suggesting that the ubiquitin ligase that promotes ARR5 proteolysis requires RUB modification for optimal activity.     

Axillary bud growth in relation to adventitious root formation in cuttings

Single-node leaf-bud cuttings of Schefflera arboricola Hayata and Stephanotis floribunda Brongn. were set and root formation, onset of axillary bud growth and plant height were measured. An increase in the number of roots in Schefflera, which was achieved with increasing cutting position on the stock plant (measured from top to base) or with increasing stem length below the node, accelerated the onset of axillary bud growth and resulted in an increase in plant height. Increasing the number of roots per cutting in Stephanotis through an increase in basal temperature also accelerated bud and shoot growth. Positional effects on root formation in Stephanotis showed no relationship with axillary bud growth and plant height. A positive relationship between number of roots per cutting and axillary bud growth was found among clones of Stephanotis . In general the results suggest that, with some exceptions, the onset of axillary bud growth is accelerated in cuttings as a result of accelerated root formation and a higher number of roots per cutting.     

Obestatin acts in brain to inhibit thirst

Derived from the same prohormone, obestatin has been reported to exert effects on food intake that oppose those of ghrelin. The obestatin receptor GPR39 is present in brain and pituitary gland. Since the gene encoding those two peptides is expressed also in those tissues, we examined further the possible actions of obestatin in vivo and in vitro. Intracerebroventricular administration of obestatin inhibited water drinking in ad libitum-fed and -watered rats, and in food-and water-deprived animals. The effects on water drinking preceded and were more pronounced than any effect on food intake, and did not appear to be the result of altered locomotor/behavioral activity. In addition, obestatin inhibited ANG II-induced water drinking in animals provided free access to water and food. Current-clamp recordings from cultured, subfornical organ neurons revealed significant effects of the peptide on membrane potential, suggesting this as a potential site of action. In pituitary cell cultures, log molar concentrations of obestatin ranging from 1.0 pM to 100 nM failed to alter basal growth hormone (GH) secretion. In addition, 100 nM obestatin failed to interfere with the stimulation of GH secretion by GH-releasing hormone or ghrelin and did not alter the inhibition by somatostatin in vitro. We conclude that obestatin does not act in pituitary gland to regulate GH secretion but may act in brain to alter thirst mechanisms. Importantly, in rats the effects of obestatin on food intake may be secondary to an action of the peptide to inhibit water drinking.     

Arabidopsis PEAPODs function with LIKE HETEROCHROMATIN PROTEIN1 to regulate lateral organ growth

In higher plants, lateral organs are usually of determinate growth. It remains largely elusive how the determinate growth is achieved and maintained. Previous reports have shown that Arabidopsis PEAPOD (PPD) proteins suppress proliferation of dispersed meristematic cells partly through a TOPLESS corepressor complex. Here, we identified a new PPD‐interacting partner, LIKE HETEROCHROMATIN PROTEIN1 (LHP1), using the yeast two‐hybrid system, and their interaction is mediated by the chromo shadow domain and the Jas domain in LHP1 and PPD2, respectively. Our genetic data demonstrate that the phenotype of ppd2 lhp1 is more similar to lhp1 than to ppd2, indicating epistasis of lhp1 to ppd2. Microarray analysis reveals that PPD2 and LHP1 can regulate expression of a common set of genes directly or indirectly. Consistently, chromatin immunoprecipitation results confirm that PPD2 and LHP1 are coenriched at the promoter region of their targets such as D3‐TYPE CYCLINS and HIGH MOBILITY GROUP A, which are upregulated in ppd2, lhp1 and ppd2 lhp1 mutants, and that PPDs mediate repressive histone 3 lysine‐27 trimethylation at these loci. Taken together, our data provide evidence that PPD and LHP1 form a corepressor complex that regulates lateral organ growth.     

Correlative inhibition of lateral bud growth in Phaseolus vulgaris L. timing of bud growth following decapitation

Summary On intact, 3-week-old plants of Phaseolus the larger bud in the axils of the primary leaves shows slow, continuous elongation growth. Release from correlative inhibition can be detected within 30 min following decapitation. When 0.1% indoleacetic acid in lanolin is applied to the decapitated stem stump, the lateral bud shows slow growth during the first 7 h, then stops completely for a further 15 h but after 2 days a further gradual increase in length is observed.The movement of ¹⁴C-labelled assimilates from the subtending primary leaf into the lateral bud increases following removal of the shoot apex. When indole acetic acid is applied to decapitated plants the ability of the buds to import ¹⁴C increases for 5–7 h and then declines to a negligible amount. Little or no radioactivity from tritiated indoleacetic acid is transported into the lateral buds of decapitated plants during the first 48 h following removal of the apex and it appears that rapid metabolism of the compound occurs in the stem tissues.     

AXR1-ECR1 and AXL1-ECR1 heterodimeric RUB-activating enzymes diverge in function in Arabidopsis thaliana

RELATED TO UBIQUITIN (RUB) modification of CULLIN (CUL) subunits of the CUL-RING ubiquitin E3 ligase (CRL) superfamily regulates CRL ubiquitylation activity. RUB modification requires E1 and E2 enzymes that are analogous to, but distinct from, those activities required for UBIQUITIN (UBQ) attachment. Gene duplications are widespread in angiosperms, and in line with this observation, components of the RUB conjugation pathway are found in multiples in Arabidopsis. To further examine the extent of redundancy within the RUB pathway, we undertook biochemical and genetic characterizations of one such duplication event- the duplication of the genes encoding a subunit of the RUB E1 into AUXIN RESISTANT1 (AXR1) and AXR1-LIKE1 (AXL1). In vitro, the two proteins have similar abilities to function with E1 C-TERMINAL-RELATED1 (ECR1) in catalyzing RUB1 activation and RUB1-ECR1 thioester formation. Using mass spectrometry, endogenous AXR1 and AXL1 proteins were found in complex with 3HA-RUB1, suggesting that AXR1 and AXL1 exist in parallel RUB E1 complexes in Arabidopsis. In contrast, AXR1 and AXL1 differ in ability to correct phenotypic defects in axr1-30, a severe loss-of-function AXR1 mutant, when the respective coding sequences are expressed from the same promoter, suggesting differential in vivo functions. These results suggest that while both proteins function in the RUB pathway and are biochemically similar in RUB-ECR1 thioester formation, they are not functionally equivalent.     

The MSG1 and AXR1 genes of Arabidopsis are likely to act independently in growth-curvature responses of hypocotyls

Growth-curvature responses of hypocotyls of Arabidopsis thaliana (L.) Heynh. were measured in double mutants between msg1 and axr1, both of which are auxin-resistant and defective in hypocotyl growth curvature induced upon unilateral application of auxin. The msg1 axr1 double mutants showed no auxin-induced growth curvature, that is, they exhibited the msg1 phenotype, though the axr1 defects were partial. Hypocotyls of both the msg1 and axr1 mutants were partially defective in second-positive phototropism, whereas the double mutants lost the response completely. When grown on vertically held agar plates, the axr1 mutant showed normal hypocotyl gravitropism and the mutation did not affect the reduced hypocotyl gravitropism of msg1. Hypocotyls of msg1 and axr1 mutants grew upward like wild-type ones when grown along an agar surface, while they grew more randomly when grown without an agar support, suggesting that axr1 hypocotyls are not completely normal in gravitropism. The extent of defects in growth orientation increased in the order: msg1 axr1 double mutants > msg1 > axr1 > wild type. The hypocotyls of these mutants showed auxin resistance in the order: msg1 axr1 > axr1 > msg1 > wild type. The msg1 mutant had epinastic leaves and axr1 had wrinkled leaves; leaves of the msg1 axr1 double mutants were epinastic and wrinkled. These results suggest that MSG1 and AXR1 act independently in separate pathways of the reactions tested in the present study. In contrast, the phenotype of the msg1 aux1 double mutants shows that AUX1 is not significantly involved in these phenomena. Received: 12 July 1998 / Accepted: 16 August 1998     

AXR1-ECR1-dependent conjugation of RUB1 to the Arabidopsis Cullin AtCUL1 is required for auxin response

Mutations in the AXR1 gene result in a reduction in auxin response and diverse defects in auxin-regulated growth and development. In a previous study, we showed that AXR1 forms a heterodimer with the ECR1 protein. This enzyme activates the ubiquitin-related protein RUB1 in vitro. Furthermore, we showed that the Skp1-Cul1/Cdc53-F-box (SCF) subunit AtCUL1 is modified by RUB1 in vivo. In this report, we demonstrate that the formation of RUB-AtCUL1 is dependent on AXR1 and ECR1 in vivo. The expression of AXR1 and ECR1 is restricted to zones of active cell division and cell elongation, consistent with their role in growth regulation. These results provide strong support for a model in which RUB conjugation of AtCUL1 affects the function of SCF E3s that are required for auxin response.     

The bHLH protein ROX acts in concert with RAX1 and LAS to modulate axillary meristem formation in Arabidopsis

During post-embryonic shoot development, new meristems are initiated in the axils of leaves. They produce secondary axes of growth that determine morphological plasticity and reproductive efficiency in higher plants. In this study, we describe the role of the bHLH-protein-encoding Arabidopsis gene REGULATOR OF AXILLARY MERISTEM FORMATION (ROX), which is the ortholog of the branching regulators LAX PANICLE1 (LAX1) in rice and barren stalk1 (ba1) in maize. rox mutants display compromised axillary bud formation during vegetative shoot development, and combination of rox mutants with mutations in RAX1 and LAS, two key regulators of axillary meristem initiation, enhances their branching defects. In contrast to lax1 and ba1, flower development is unaffected in rox mutants. Over-expression of ROX leads to formation of accessory side shoots. ROX mRNA accumulates at the adaxial boundary of leaf and flower primordia. However, in the vegetative phase, axillary meristems initiate after ROX expression has terminated, suggesting an indirect role for ROX in meristem formation. During vegetative development, ROX expression is dependent on RAX1 and LAS activity, and all three genes act in concert to modulate axillary meristem formation.     

Antiegressin acts late in the intracellular growth cycle of Trypanosoma cruzi to inhibit parasite egress

Trypanosoma cruzi is the causative agent of Chagas disease, which is characterized by acute and chronic phases. During the former, parasitemia rises dramatically, then decreases significantly during the chronic phase. Immune mechanisms responsible for the parasitemia reduction have not been thoroughly elucidated. The goal of the present study was to further characterize the immune response during chronic infection. Previously, we described antiegressin, an antibody in sera from chronically infected mice. The in vitro presence of antiegressin inhibits parasite egress from infected host cells. Antiegressin appears by day 14 of an in vivo infection and is maintained through at least day 280 postinfection. The in vitro functional activity of antiegressin is initiated late in the 4-6 days intracellular growth cycle of T. cruzi; antiegressin may be added at day 4, inhibiting parasite release at day 5. Immunocytochemical staining using antineuraminidase demonstrates the presence of mature parasites inside host BALB/c fibroblasts grown in the presence of antiegressin. These results demonstrate the ability of antiegressin to inhibit emergence of developmentally mature trypomastigotes from infected host cells late in their intracellular growth cycle. We believe this antibody plays an important and novel role in achieving the low-parasitemia characteristic of chronic Chagas disease.