Mutations in LIKE HETEROCHROMATIN PROTEIN 1 affect flowering time and plant architecture in Arabidopsis.

In plants, recent studies have demonstrated links between the regulation of developmental processes and chromatin dynamics and organisation. Analysis of new mutations affecting overall plant architecture, leaf development and flowering time in Arabidopsis has allowed us to clone and characterise LHP1, the Drosophila heterochromatin protein 1 (HP1) homologue. LHP1 has the chromo and chromo shadow domains central to the function of animal proteins. Yeast two hybrid studies and in planta deletion experiments suggest similar modes of action in plants and animals via homodimer formation. In vivo localisation experiments revealed a specific subnuclear protein distribution in foci throughout the nucleus. Our data suggest that LHP1 may act as a main regulator of gene expression in plants, through formation of heterochromatin-like repressive complexes, to control developmental pathways involved in organ and cell size, and the vegetative to reproductive phase transition.

Quantitative control of inflorescence formation in impatiens balsamina

We analyzed the process of inflorescence formation in Impatiens balsamina by studying the architecture of the plant under different photoperiod treatments. Floral reversion under noninductive conditions in this species is caused by the lack of persistence of the induced state in the leaf. This can be used to control the amount of inductive signal and to examine its quantitative influence on morphological changes in the plant. The floral transition was characterized by a continuum of variation at the level of meristem identity, primordium initiation, and floral organ identity. This continuum was enhanced during reversion, suggesting that the establishment of a continuum partly reflects limiting amounts of inductive signal exported from the leaf to the meristem. The transcription patterns of two homologs of genes involved in the control of floral meristem identity, Imp-FLO and Imp-FIM, were similar in terminal and axillary flowers and may be associated with the continuum exhibited by I. balsamina. By analyzing the fate of axillary meristem primordia initiated before and after the beginning of the inductive period, we showed that de novo initiation of axillary meristem primordia by the evoked meristem is not required and that primordia initiated before evocation can adopt different fates, depending on the amount of inductive signal. The influence of age and/or position on primordium responsiveness to the inductive signal is discussed.

Transcription pattern of a FIM homologue in Impatiens during floral development and reversion.

Flowering and reversion in Impatiens are characterised by gradual transitions of organ identity and constitute a unique system for the molecular and physiological study of floral organogenesis. The authors have isolated an Impatiens homologue of the FIM gene of Antirrhinum (UFO in Arabidopsis), Imp-FIM, and analysed its expression in three states of the terminal meristem: vegetative, floral, and reverted. In floral meristems, Imp-FIM transcription is associated with petal identity, as in Antirrhinum and Arabidopsis, but this is achieved through a novel transcription pattern, characterised by a high level of transcript within petal primordia. This novel transcription pattern could contribute to the more diffuse boundaries between organ types in Impatiens. In vegetative meristems, Imp-FIM is expressed in the axils of leaf primordia which are arranged in a spiral. A similar pattern is observed in reverted meristems in which leaf primordia are initiated in a whorled arrangement. This result indicates that the maintenance of floral phyllotaxis is not associated with a specific pattern of Imp-FIM transcription. Transcription of Imp-FIM in a non-reverting line is no different from that in the reverting line. Therefore, the lack of floral commitment in the reverting line does not seem to be responsible for Imp-FIM transcription within petals. The novel transcription pattern in petals, together with features of Impatiens that are reminiscent of fim and ufo mutant phenotypes suggest an evolutionary divergence for Imp-FIM regulation in this species.

The induction and maintenance of flowering in Impatiens.

The mechanisms that establish the floral meristem are now becoming clearer, but the way in which flowering is maintained is less well understood. Impatiens balsamina provides a unique opportunity to address this question because reversion to vegetative growth can be obtained in a predictable way by transferring plants from inductive to non-inductive conditions. Following increasing amounts of induction, reversion takes place at progressively later stages of flower development. Partial flower induction and defoliation experiments show that a floral signal is produced in the cotyledon in response to inductive conditions and that this signal progressively diminishes after transfer to non-inductive conditions, during reversion. Therefore reversion in Impatiens is most likely due to the failure of leaves to become permanent sources of inductive signal in addition to the lack of meristem commitment to flowering. Analysis of the expression of the Impatiens homologues of the meristem identity genes floricaula and squamosa indicates that a change in floricaula transcription is not associated with the establishment or maintenance of the floral meristem in this species. Squamosa transcription is associated with floral development and petal initiation, and is maintained in existing petal or petaloid primordia even after the meristem has reverted. However, it is not expressed in the reverted meristem, in which leaves are initiated in whorled phyllotaxis and without axillary meristems, both characteristics usually associated with the floral meristem. These observations show that squamosa expression is not needed for the maintenance of these floral characters. The requirement for the production of the floral signal in the leaf during the process of flower development may reflect an additional function separate to that of squamosa activation; alternatively the signal may be required to ensure continued transcriptional activation in the meristem.

Functional analysis of the tobacco Tnt1 retrotransposon.

Retroelements represent by far the largest and most widespread class of mobile genetic elements. Representative of several classes of retrotransposons have been characterized in a broad range of plant species, but only a few of them have been shown to be active. Among these, the tobacco Tnt1 retrotransposon has been isolated after insertion mutagenesis and is one of the very few to be transcriptionally active. Tnt1 expression is strongly regulated in a tissue-specific and developmental manner. Moreover, Tnt1 expression is induced by a range of biotic or abiotic elicitors, which all have in common the ability to induce the plant defense response. Regulatory sequences involved in this elicitor-mediated induction have been located in the LTR U3 region. The link between Tnt1 activation and the plant defense response might represent an example of the involvement of transposable elements in genome restructurations needed in response to environmental fluctuations such as pathogen attacks.

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia.

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after gamma-ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 bp insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 bp terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5' and 3' ends of dTnp1 together with a perfect palindrome located after the 5' inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.

Effects of Tnt1 tobacco retrotransposon insertion on target gene transcription.

The effects of Tnt1 retrotransposon insertion on nitrate reductase (NR) gene transcription have been analyzed in three NR-deficient insertional, mutants of Nicotiana tabacum. In the three mutants, named h9-Nia4, h9-Nia5 and h9-Nia6, Tnt1 was inserted into exon 3, exon 2 and exon 1 of the nia2 NR alloallelle, respectively. The mutants h9-Nia4 and h9-Nia6, which contained Tnt1 insertions that were oriented opposite to the direction of nia2 gene transcription, expressed chimaeric nia2-Tnt1 RNAs, respectively 12 kb and 10 kb long. The size observed in h9-Nia6 was close to the expected size for a full-length hybrid transcript starting and ending under the control of nia2 signals (about 9 kb). The larger transcript found in h9-Nia4 was shown to be due to a failure to splice the nia2 intron 2. The mutant h9-Nia5, which contained a Tnt1 insertion oriented in parallel with the direction of nia2 transcription expressed two truncated nia2-Tnt1 RNAs, 2 kb and 6.7 kb long. These transcripts arose from termination in the long terminal repeats (LTRs) of Tnt1. Since no full-length hybrid RNA was detected, we suggest that Tnt1 carries efficient termination signals, which are more efficiently recognized in the 3' LTR than in the 5' LTR.

Specific expression of the tobacco Tnt1 retrotransposon in protoplasts.

The Tnt1 transposable element of tobacco belongs to the retrotransposon family and shares the structural features of viral retroelements including two long terminal repeats (LTRs) which are known to contain promoter regions. We show that two Tnt1 RNAs of 5.2 and 6.5 kb can be found. The 5.2 kb RNA matches with the size of the Tnt1 elements so far isolated (5.3 kb), whilst the evidence suggests that the 6.5 kb RNA could be a chimaeric RNA initiated in a gene in which Tnt1 has inserted. The Tnt1 5.2 kb RNA starts in the LTR, and the LTR can promote the expression of a translational LTR-beta-glucuronidase (GUS) fusion at a high level in transient expression assays. The Tnt1 5.2 kb RNA and the LTR-GUS fusion of transgenic tobacco plants are specifically expressed in leaf-derived protoplasts whereas they are not expressed in leaf tissue. The 5.2 kb RNA is also transcribed at low levels in roots. This RNA is induced after 2 h of maceration in the protoplast isolation medium, and its level declines rapidly after protoplast isolation. The induction requires only the presence of cell wall hydrolases, and is independent of wounding and plasmolysis. The induction of Tnt1 expression is not mediated by typical oligosaccharide elicitors released from the cell wall known to mediate defense gene responses. Tnt1 transcription features provide a first example of tissue culture-induced mutagenesis in plants and a molecular basis for some of the somaclonal variation events.

Nitrate Reductase mRNA Regulation in Nicotiana plumbaginifolia Nitrate Reductase-Deficient Mutants.

Light and substrate regulation of nitrate reductase (NR) expression were compared in wild type and mutant lines of Nicotiana plumbaginifolia. Mutants affected in the NR structural gene (nia) or in the biosynthesis of the NR molybdenum cofactor (cnx) were examined. nia mutants expressing a defective apoenzyme, as well as cnx mutants, overexpressed NR mRNA, whereas nia mutants devoid of detectable NR protein had reduced or undetectable NR mRNA levels. Diurnal fluctuations of NR mRNA were specifically abolished in nia and cnx mutants, suggesting that the integrity of NR catalytic activity is required for the expression of diurnal oscillations. Unlike some fungal mutants, the nia and cnx mutants examined retained nitrate inducibility of NR expression. The possibility of autogenous control of NR expression in higher plants is discussed.