BpMADS4 has a central role in inflorescence initiation in silver birch (Betula pendula).

Acceleration of flowering would be beneficial for breeding trees with a long juvenile phase; conversely, inhibition of flowering would prevent the spread of transgenes from the genetically modified trees. We have previously isolated and characterized several MADS genes from silver birch (Betula pendula Roth). In this study, we investigated the more detailed function of one of them, BpMADS4, a member of the APETALA1/FRUITFULL group of MADS genes. The expression of BpMADS4 starts at very early stage of the male and female inflorescence development and the activity is high in the apex of the developing inflorescence. Later, some expression is detected in the bracts and in the flower initials. Ectopic expression of BpMADS4 accelerates flowering dramatically in normally flowering clones and also in the early-flowering birch clone, in which the earliest line flowered about 11 days after rooting, when the saplings were only 3 cm high. The birches transformed with the BpMADS4 antisense construct showed remarkable delay in flowering and the number of flowering individuals was reduced. Two of the transformed lines did not show any signs of flower development during our 2-year study, whereas all the control plants formed inflorescences within 107 days. Our results show that BpMADS4 has a critical role in the initiation of birch inflorescence development and that BpMADS4 seems to be involved in the transition from vegetative to reproductive development. Therefore, BpMADS4 provides a promising tool for the genetic enhancement of forest trees.

Functional characterization of SEPALLATA3 and AGAMOUS orthologues in silver birch.

The development of flowers is regulated by a complex network of transcriptional activators and repressors, many of which belong to the MADS box gene family. In this study, we describe two MADS box genes of silver birch (Betula pendula Roth), BpMADS1 and BpMADS6, which are similar to SEPALLATA3 and AGAMOUS in Arabidopsis thaliana, respectively. In situ hybridization showed that BpMADS1 was expressed in the inflorescence meristem at a very early stage, but not later. Both genes were expressed in developing carpels, ovules and stamens but not in tepals or scales. Ectopic expression of BpMADS1 in Arabidopsis resulted in a reduced number of floral organs or whole whorls and in petaloid or carpelloid sepals, a phenotype reminiscent of that of fil mutants. 35S::BpMADS6 caused very early flowering in Arabidopsis. In tobacco, both 35S::BpMADS1 and 35S::BpMADS6 accelerated flowering and, in addition, 35S::BpMADS6 caused changes in sepals and petals. In some transgenic birch plants, 35S::BpMADS1 antisense resulted in the development of both male and female organs in the axil of a single bract and in a change of some inflorescences into vegetative shoots. In two plants, either 35S::BpMADS6 sense or antisense constructs resulted in an increase in the number of tepals and in complete lack of stamens in some male inflorescences. These results suggest that BpMADS1 participates both in inflorescence and in flower formation and BpMADS6 participates in flower formation and that they are functional homologues to SEPALLATA3 and AGAMOUS, respectively.

A new SBP-box gene BpSPL1 in silver birch (Betula pendula).

The SBP-box gene family represents a group of plant-specific genes encoding putative transcription factors. Thus far, SBP-domain protein binding sites have been found in the promoters of Arabidopsis APETALA1 and Antirrhinum SQUAMOSA. A putative SBP-domain binding element has been observed in the promoter of BpMADS5, a close homologue of Arabidopsis FRUITFULL in silver birch (Betula pendula). A novel SBP-box gene from birch named BpSPL1 has been cloned and characterized. The nucleotide sequence of BpSPL1 is similar to Antirrhinum SBP2 and Arabidopsis SPL3, apart from the unique finding that BpSPL1 does not contain an intron typical to all other known SBP-box genes studied thus far. According to Northern blot analysis, BpSPL1 is expressed in birch inflorescences as well as in shoots and leaves. Studies using electrophoretic mobility shift assay demonstrate that there are nuclear proteins in birch inflorescences which specifically bind to the SBP binding element of the promoter of BpMADS5. BpSPL1 expressed in Escherichia coli also specifically binds to this element. According to Southern blot analysis, there are at least two SBP-box genes in birch. The results suggest that SBP-box genes are involved in the regulation of flower development in birch.

Glutamine synthetase isozymes in germinating barley seeds.

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of ammonia assimilation in higher plants. In the present study the subunit composition and localization of GS in germinating barley (Hordeum vulgare) seed have been clarified. Analysis of the GS polypeptide composition by immunoblotting revealed two different polypeptides. A and B, with a molecular mass of 42 and 40 kDa, respectively. In the scutellum subunit A was already present in the ungerminated seed and remained unchanged, whereas subunit B appeared on day 2 and increased about 5-fold during germination. Polypeptide B also appeared later during germination in the aleurone layer, roots and weakly in the etiolated shoots. By immunogold microscopy, GS was detected in the scutellum and the aleurone layer of barley seeds during germination. Subcellular localization of GS on ultrathin cryosections showed that a cytosolic isozyme was present in the scutellum. Our study confirms that only a cytosolic GS is expressed in barley seed, and its subunit composition changes during germination.