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.     

Three MADS-box genes similar to APETALA1 and FRUITFULL from silver birch (Betula pendula)

Despite intensive research on genetic regulation of flower development there are still only a few studies on the early phases of this process in perennial plants like trees. The aim of this study has been to identify genes that regulate early stages of inflorescence development in silver birch ( Betula pendula Roth) and to follow the expression of these genes during development of the unisexual birch inflorescences. Here we describe the cloning and characterization of 3 cDNAs representing MADS-box genes designated BpMADS3, BpMADS4 and BpMADS5, all belonging to the AP1/SQUA group of plant MADS-box genes. According to RNA blot analysis, all 3 genes are active during the development of both male and female inflorescences. However, differences in patterns of expression suggest that they play different roles. BpMADS3 is most similar in sequence to AP1 and SQUA, but it seems to have the highest expression at late developmental stages. BpMADS4 is most similar in sequence to the Arabidopsis gene FRUITFULL , but is expressed, in addition to developing inflorescences, in shoots and roots. BpMADS5 is also similar to FRUITFULL; its expression seems to be inflorescence-specific and continues during fruit development. Ectopic expression of either BpMADS3, BpMADS4 or BpMADS5 with the CaMV 35S promoter in tobacco results in extremely early flowering. All of these birch genes seem to act early during the transition to reproductive phase and might be involved in the determination of the identity of the inflorescence or flower meristem. They could apparently be used to accelerate flowering in various plant species.     

Influence of Nitrogen,Phosphorus, and Potassium Fertilization on Flowering and Expression of Flowering-Associated Genes in White Birch (<Emphasis Type="Italic">Betula platyphylla</Emphasis> Suk.)

Fertilization treatments can promote the growth and development of tree plants. In the present study, we evaluated the combined effects of different doses of nitrogen, phosphorous, and potassium on the blossoming and the expression of five flowering-associated genes, including BpMADS1, BpMADS3, BpMADS5, BpSPL1, and BpSPL2. The results showed that balanced NPK (nitrogen, phosphorous, and potassium) fertilization cannot only promote the transition from juvenility to maturity but also can increase the inflorescence production of white birch. Furthermore, some fertilizing treatments not only increase the expression levels of all the five flowering-associated genes, but also change their expression patterns during the growth season. These results indicated that NPK fertilization have remarkable effects on flowering of white birch.     

Prevention of the flowering of a tree,silver birch

Genetic modification of trees presents great advantages but it is hampered by the possible spread of introduced genes to native populations. However, the spread would be prevented if the modified trees would be sterile. We have previously shown that the induction of sterility by the prevention of flowering is possible in tobacco and Arabidopsis by introducing a gene construct composed of the ribonuclease gene BARNASE ligated to the flower-specific promoter of the birch gene BpMADS1. In the present study, we test this gene construct in silver birch (Betula pendula Roth). When this gene construct was introduced into very early-flowering birch clones, 81 kanamycin resistant lines were obtained. In 38 lines, the vegetative development was disturbed, e.g., the leaves were small and the plants were short and bushy or the growth of plants was weak. More importantly, in 7 other lines no male inflorescences formed or they aborted early. If male inflorescences were formed, they did not contain any stamens. The initial growth of these lines was similar to the non-transgenic control lines. Later, however, the growth of the non-flowering lines differed from that of the controls in showing some dichotomic branching and a reduced number of branches. Preliminary results showed that the gene construct can prevent the development of female inflorescences as well. The results show clearly that BpMADS1::BARNASE can prevent the flowering in a tree but the prevention of flowering may cause some side effects. Studies with ordinary birch clones will show whether the side effects are a property of the early flowering clones or all birches.     

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.     

Significance of consensus CYC-binding sites found in the promoters of both ChCYC and ChRAD genes in Chirita heterotricha (Gesneriaceae)

CYC-like genes are widely conserved in controlling floral dorsoventral asymmetry (zygomorphy) through persistent expression in corresponding domains in core eudicots. To understand how CYC-like gene expression is maintained during flower development, we selected Chirita heterotricha as a material and isolated the promoter sequences of the ChCYCIC and ChCYCID genes, homologs of CYC, by inverse polymerase chain reaction. Further promoter analyses led to the identification of a putative cis-regulatory element in each promoter matching the consensus DNA binding site for Antirrhinum CYC protein: GGCCCCTC at-165 for ChCYC1C, and GGCCCCCC at-163 for ChCYCID. This indicates that both the ChCYCIC and ChCYC1D genes have probably evolved autoregulatory loops to sustain their expression in developing flowers. We also isolated the coding and promoter sequences of the ChRAD gene, a homolog of Antirrhinum RAD. Promoter analysis showed that the ChRAD gene promoter also contained a putative CYC-binding site (GGCCCAC at -134). Therefore, ChRAD is likely a direct target of the ChCYC1 genes, which is similar to Antirrhinum RAD. These results imply that the establishment of floral zygomorphy in Chirita may have been achieved by the evolution of an autoregulatory loop for CYC-like genes,which was probably accompanied by simultaneous co-option of the RAD-like gene into their regulatory network.     

Functional conservation of PISTILLATA activity in a pea homolog lacking the PI motif

Current understanding of floral development is mainly based on what we know from Arabidopsis (Arabidopsis thaliana) and Antirrhinum majus. However, we can learn more by comparing developmental mechanisms that may explain morphological differences between species. A good example comes from the analysis of genes controlling flower development in pea (Pisum sativum), a plant with more complex leaves and inflorescences than Arabidopsis and Antirrhinum, and a different floral ontogeny. The analysis of UNIFOLIATA (UNI) and STAMINA PISTILLOIDA (STP), the pea orthologs of LEAFY and UNUSUAL FLORAL ORGANS, has revealed a common link in the regulation of flower and leaf development not apparent in Arabidopsis. While the Arabidopsis genes mainly behave as key regulators of flower development, where they control the expression of B-function genes, UNI and STP also contribute to the development of the pea compound leaf. Here, we describe the characterization of P. sativum PISTILLATA (PsPI), a pea MADS-box gene homologous to B-function genes like PI and GLOBOSA (GLO), from Arabidopsis and Antirrhinum, respectively. PsPI encodes for an atypical PI-type polypeptide that lacks the highly conserved C-terminal PI motif. Nevertheless, constitutive expression of PsPI in tobacco (Nicotiana tabacum) and Arabidopsis shows that it can specifically replace the function of PI, being able to complement the strong pi-1 mutant. Accordingly, PsPI expression in pea flowers, which is dependent on STP, is identical to PI and GLO. Interestingly, PsPI is also transiently expressed in young leaves, suggesting a role of PsPI in pea leaf development, a possibility that fits with the established role of UNI and STP in the control of this process.     

Prevention of flower formation in dicotyledons

Prevention of flower formation is important, for example for preventing the spread of transgenes from genetically modified plants or the spread of non-native species, for increasing vegetative growth or preventing the formation of allergenic pollen. The aim of this study was to determine whether flowering of dicotyledonous plants can be prevented by genetic manipulation without harmful effects on vegetative growth. Here we describe isolation of the BpMADS1 gene (similar to SEP3, formerly AGL9) from birch and show that it is expressed only in the inflorescences. In tobacco and Arabidopsis, the expression of BpMADS1::GUS was also virtually inflorescence-specific. Transgenic tobacco and Arabidopsis containing a BpMADS1::BARNASE construct grew well. In one tobacco line the formation of the inflorescence was completely prevented; in several other lines the flowers lacked stamens and carpels and therefore were sterile. The final dry weights of the shoots of the sterile tobacco lines were 140–200% of those of controls. In Arabidopsis, some of the transgenic lines containing the BpMADS1::BARNASE construct formed inflorescences. Some of these lines formed never flowers and some others formed occasionally single fertile flowers. Some other lines did not form inflorescences, but formed up to about one hundred leaves, even in long-day conditions. These results suggest that formation of flowers or inflorescences in widely different dicotyledonous plants could be prevented using the BpMADS1::BARNASE construct and that prevention of flowering may lead to increased vegetative mass.     

Prevention of flower development in birch and other plants using a BpFULL1::BARNASE construct

The prevention of flower formation is important for avoiding the spread of transgenes from genetically modified plants into wild populations. Moreover, the resources not expended for the generation of flowers and fruits might be allocated to increased vegetative growth. We have been developing methods for preventing flower formation in silver birch (Betula pendula), a tree species of considerable economical importance in the boreal region. Here we study the suitability of the promoter of BpFRUITFULL-LIKE1 (BpFULL1, formerly BpMADS5) for tissue-specific ablation of inflorescences in Arabidopsis, tobacco and birch. With all these species, the development of inflorescences was successfully prevented. The results show that the BpFULL1::BARNASE construct has potential biotechnological applications in different plant species.This revised version was published online in March 2005 because the name of the second author (M. Hassinen) was missing.