Cytokinin signaling regulates cambial development in poplar.

Although a substantial proportion of plant biomass originates from the activity of vascular cambium, the molecular basis of radial plant growth is still largely unknown. To address whether cytokinins are required for cambial activity, we studied cytokinin signaling across the cambial zones of 2 tree species, poplar (Populus trichocarpa) and birch (Betula pendula). We observed an expression peak for genes encoding cytokinin receptors in the dividing cambial cells. We reduced cytokinin levels endogenously by engineering transgenic poplar trees (P. tremula x tremuloides) to express a cytokinin catabolic gene, Arabidopsis CYTOKININ OXIDASE 2, under the promoter of a birch CYTOKININ RECEPTOR 1 gene. Transgenic trees showed reduced concentration of a biologically active cytokinin, correlating with impaired cytokinin responsiveness. In these trees, both apical and radial growth was compromised. However, radial growth was more affected, as illustrated by a thinner stem diameter than in WT at same height. To dissect radial from apical growth inhibition, we performed a reciprocal grafting experiment. WT scion outgrew the diameter of transgenic stock, implicating cytokinin activity as a direct determinant of radial growth. The reduced radial growth correlated with a reduced number of cambial cell layers. Moreover, expression of a cytokinin primary response gene was dramatically reduced in the thin-stemmed transgenic trees. Thus, a reduced level of cytokinin signaling is the primary basis for the impaired cambial growth observed. Together, our results show that cytokinins are major hormonal regulators required for cambial development.

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.

The expression and promoter specificity of the birch homologs for PISTILLATA/GLOBOSA and APETALA3/DEFICIENS.

B-function genes determine the identity of petals and stamens in the flowers of model plants such as Arabidopsis and Antirrhinum. Here, we show that a putative B-function gene BpMADS2, a birch homolog for PISTILLATA, is expressed in stamens and carpels of birch inflorescences. We also present a novel birch gene BpMADS8, a homolog for APETALA3/DEFICIENS, which is expressed in stamens. Promoter-GUS analysis revealed that BpMADS2 promoter is active in the receptacle of Arabidopsis flower buds while BpMADS8 promoter is highly specific in mature stamens. BpMADS2 promoter::BARNASE construct prevented floral organ development in Arabidopsis and tobacco. In birch, inflorescences with degenerated stamens and carpels were obtained. BpMADS8::BARNASE resulted in degeneration of stamens in Arabidopsis and birch causing male sterility. In tobacco, only sepals were developed instead of normal flowers. The results show that the BpMADS2::BARNASE construct can be used to specifically disrupt floral organ development in phylogenetically distant plant species. The stamen-specific promoter of BpMADS8 is a promising tool for biotechnological applications in inducing male sterility or targeting gene expression in the late stamen development.

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.

Effect of parent genotype on somatic embryogenesis in Scots pine (Pinus sylvestris).

Controlled crosses of seven Scots pine (Pinus sylvestris L.) trees produced 49 families that included both reciprocals and selfings. Embryogenic cultures were initiated from immature megagametophytes and after 6 months in maintenance culture, mature somatic embryos were produced from the surviving 166 lines. The effect of parent genotypes on the cultures was evaluated at initiation of the tissue culture period, after 6 months in maintenance culture and at embryo maturation. The effect of the maternal parent was most pronounced at culture initiation. After 6 months in tissue culture, the maternal effect had decreased and the effects of both parents were significant. By the somatic embryo maturation stage, the maternal effect was still considerable but the paternal effect was no longer detectable. There was little correlation between the ranking of mothers and fathers, indicating that the maternal effect was caused by factors other than the paternal effect. No mother x father interaction was found, indicating that mothers successful at initiation and after 6 months in tissue culture, pollinated by any of the successful fathers, produced somatic lines and mature somatic embryos.