茶树花发育MADS-box转录因子CsGLO1、CsGLO2与CsAG之间的互作关系研究

利用酵母双杂交方法和双分子荧光互补技术（BiFC）研究了茶树（Camellia sinensis（L.）O.Kuntze）花发育MADS-box的B类转录因子CsGLO1、CsGLO2与C类转录因子CsAG间的互作关系及其可能发生互作的亚细胞定位。通过构建5个酵母表达载体,利用酵母单杂交实验检测了3个蛋白的转录激活活性,并通过酵母双杂交实验分析了蛋白间的互作关系。结果显示,3个蛋白均无转录激活活性,且两两之间可发生相互作用。进一步构建6个BiFC表达载体,采用压力注射法瞬时浸染烟草（Nicotiana benthamiana L.）叶表皮细胞,并利用激光共聚焦显微镜观察荧光信号,结果表明茶树B类CsGLO与C类CsAG蛋白可在植物活细胞内形成同源和异源二聚体,并具有在细胞质中发生互作的特定模式。本研究可为利用分子生物学技术抑制茶树“花果同现”现象提供理论依据。     

Cloning and characterization of cellulose synthase-like gene, PtrCSLD2 from developing xylem of aspen trees

Genetic improvement of cell wall polymer synthesis in forest trees is one of the major goals of forest biotechnology that could possibly impact their end product utilization. Identification of genes involved in cell wall polymer biogenesis is essential for achieving this goal. Among various candidate cell wall-related genes, cellulose synthase-like D (CSLD) genes are intriguing due to their hitherto unknown functions in cell wall polymer synthesis but strong structural similarity with cellulose synthases (CesAs) involved in cellulose deposition. Little is known about CSLD genes from trees. In the present article PtrCSLD2, a first CSLD gene from an economically important tree, aspen (Populus tremuloides) is reported. PtrCSLD2 cDNA was isolated from an aspen xylem cDNA library and encodes a protein that shares 90% similarity with Arabidopsis AtCSLD3 protein involved in root hair tip growth. It is possible that xylem fibers that also grow by intrusive tip growth may need expression of PtrCSLD2 for controlling the length of xylem fibers, a wood quality trait of great economical importance. PtrCSLD2 protein has a N-terminal cysteine-rich putative zinc-binding domain; eight transmembrane domains; alternating conserved and hypervariable domains; and a processive glycosyltransferases signature, D, D, D, QXXRW; all similar to aspen CesA proteins. However, PtrCSLD2 shares only 43-48% overall identity with the known aspen CesAs suggesting its distinct functional role in cell wall polymer synthesis perhaps other than cellulose biosynthesis. Based on Southern analysis, the aspen CSLD gene family consists of at least three genes and this gene copy estimate is supported by phylogenetic analysis of available CSLDs from plants. Moreover, gene expression studies using RT-PCR and in situ mRNA hybridization showed that PtrCSLD2 is expressed at a low level in all aspen tissues examined with a slightly higher expression level in secondary cell wall-enriched aspen xylem as compared to primary cell wall enriched tissues. Together, these observations suggest that PtrCSLD2 gene may be involved in the synthesis of matrix polysaccharides that are dominant in secondary cell walls of poplar xylem. Future molecular genetic analyses will clarify the functional significance of CSLD genes in the development of woody trees.     

Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid

In our previous studies, we identified four DEFICIENS (DEF)-like genes and one GLOBOSA (GLO)-like gene involved in floral organ development in Phalaenopsis equestris. Revealing the DNA binding properties and protein-protein interactions of these floral homeotic MADS-box protein complexes (PeMADS) in orchids is crucial for the elucidation of the unique orchid floral morphogenesis. In this study, the interactome of B-class PeMADS proteins was assayed by the yeast two-hybrid system (Y2H) and glutathione S-transferase (GST) pull-down assays. Furthermore, the DNA binding activities of these proteins were assessed by using electrophoretic mobility shift assay (EMSA). All four DEF-like PeMADS proteins interacted individually with the GLO-like PeMADS6 in Y2H assay, yet with different strengths of interaction. Generally, the PeMADS3/PeMADS4 lineage interacted more strongly with PeMADS6 than the PeMADS2/PeMADS5 lineage did. In addition, independent homodimer formation for both PeMADS4 (DEF-like) and PeMADS6 (GLO-like) was detected. The protein-protein interactions between pairs of PeMADS proteins were further confirmed by using a GST pull-down assay. Furthermore, both the PeMADS4 homodimer and the PeMADS6 homodimer/homomultimer per se were able to bind to the MADS-box protein-binding motif CArG. The heterodimeric complexes PeMADS2-PeMADS6, PeMADS4-PeMADS6 and PeMADS5-PeMADS6 showed CArG binding activity. Taken together, these results suggest that various complexes formed among different combinations of the five B-class PeMADS proteins may increase the complexity of their regulatory functions and thus specify the molecular basis of whorl morphogenesis and combinatorial interactions of floral organ identity genes in orchids.     

Vegetative and generative dispersal capacity of field released transgenic aspen trees

Transfer of genes by pollen or wind-dispersed seed is considered a main potential risk when field release experiments with transgenic trees are initiated. In Germany, the first release experiment with genetically transformed trees was initiated in 1996. To ensure that the transgenic trees remained in the vegetative phase, the duration of the experiment was limited to 5 years. In total, 457 1-year-old trees including eight transgenic aspen lines carrying either the 35S- rolC or the rbcS- rolC gene construct, and three control clones were transferred to the field. In 1998 and 2000, 12 plants of transgenic lines all carrying the 35S- rolC gene construct formed female flower buds. Furthermore, one young aspen plant identified as a root sucker was observed in 1999 followed by an increasing number of root suckers derived from transgenic and non-transgenic trees in 2000 and 2001. In 2001, the last year of the field trial, 15 root suckers were detected outside the field. In total, 234 root suckers were harvested in 2000 and 2001 and analysed for their transgenic status. More than half of the roots suckers investigated showed the presence of the rbcS- rolC gene construct. We concluded that in addition to the widely accepted generative propagation, vegetative dispersal capacity of transgenic perennial plants is also important and must be included in risk assessment studies.     

Ovule-specific MADS-box proteins have conserved protein-protein interactions in monocot and dicot plants

OsMADS13 is a rice MADS-box gene that is specifically expressed in developing ovules. The amino acid sequence of OsMADS13 shows 74% similarity to those of FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, the products of two MADS-box genes that are necessary and sufficient to determine ovule identity in Petunia. To assess whether OsMADS13, the putative rice ortholog of FBP7 and FBP11, has an equivalent function, several analyses were performed. Ectopic expression of FBP7 and FBP11 in Petunia results in ectopic ovule formation on sepals and petals. Here we show that ectopic expression of OsMADS13 in rice and Arabidopsis does not result in the formation of such structures. Furthermore, ectopic expression of FBP7 and FBP11 in Arabidopsis also fails to induce ectopic ovule formation. To determine whether protein-protein interactions involving putative class D MADS-box proteins have been conserved, yeast two-hybrid assays were performed. These experiments resulted in the identification of three putative partners of OsMADS13, all of them encoded by AGL2-like genes. Interestingly the Petunia FBP7 protein also interacts with AGL2-like proteins. The evolutionary conservation of the MADS-box protein partners of these ovule-specific factors was confirmed by exchange experiments which showed that the protein partners of OsMADS13 interact with FBP7 and vice versa.     

MADS-box gene expression in lateral primordia,meristems and differentiated tissues of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> roots

Although MADS-box genes involved in flower and fruit development have been well characterized, the function of MADS-box genes expressed in vegetative structures has yet to be explored. At least seven members of this family are grouped in clades of genes that are preferentially expressed in roots of Arabidopsis thaliana (L.) Heynh.. We report here the cloning of the AGL21 MADS-box gene, which belongs to the ANR1 clade, and the mRNA in situ expression patterns of this and two other root MADS-box genes. AGL17 appears to be a lateral root cap marker in the root tip, and towards the elongation zone this gene is expressed in the epidermal cells. AGL21 is highly expressed in lateral root primordia and it has a punctate expression pattern in the primary root meristem. AGL12 also has a punctate expression pattern in the primary root meristem. AGL12 and AGL21 are also expressed in the central cylinder of differentiated roots and both are expressed in developing embryos. This study, combined with previous phylogenetic analyses, indicates that these MADS-box genes may play distinct regulatory roles during root development.     

Alteration of floral organ identity in rice through ectopic expression of<Emphasis Type="Italic"> OsMADS16</Emphasis>

We used a transgenic approach and yeast two-hybrid experiments to study the role of the rice ( Oryza sativa L.) B-function MADS-box gene, OsMADS16. Transgenic rice plants were generated that ectopically expressed OsMADS16 under the control of the maize ( Zea mays L.) ubiquitin1 promoter. Microscopic observations revealed that the innermost-whorl carpels had been replaced by stamen-like organs, which resembled the flowers of the previously described Arabidopsis thaliana (L.) Heynh. mutation superman as well as those ectopically expressing the AP3 gene. These results indicate that expression of OsMADS16 in the innermost whorl induces stamen development. Occasionally, carpels had completely disappeared. In addition, ectopic expression of OsMADS16 enhanced expression of OsMADS4, another B-function gene, causing superman phenotypes. In the yeast two-hybrid system, OsMADS16 did not form a homodimer but, rather, the protein interacted with OsMADS4. OsMADS16 also interacted with OsMADS6 and OSMADS8, both of which are homologous to SEPALLATA proteins required for the proper function of class-B and class-C genes in Arabidopsis. Based on the gene expression pattern and our yeast two-hybrid data, we discuss a quartet model of MADS-domain protein interactions in the lodicule and stamen whorls of rice florets.     

Cloning and characterization of SOC1 homologs in barley (Hordeum vulgare) and their expression during seed development and in response to vernalization

A number of genes are involved in the vernalization pathway, such as VRN1, VRN2 and VRN3/FT1, whose function has been studied in barley and wheat. However, the function of the flowering and vernalization integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) has not been well studied in Triticeae, and particularly in barley. Herein, we cloned and characterized two barley SOC1-like homologs, HvSOC1-like1 and HvSOC1-like2. Primary sequence analysis of the predicted HvSOC1-like1 and HvSOC1-like2 proteins showed that they are members of the type II MADS-box protein family. Phylogenetic analysis placed the predicted proteins with other SOC1 and SOC1-like proteins from different species neighboring those from other cereal plant species. Primary and secondary structures of the predicted proteins are conserved to each other and more distant to the recently identified barley ODDSOC1 proteins. Genomic organization of HvSOC1-like1 is very similar to the Arabidopsis and Brachypodium SOC1 genes and localized in highly syntenic chromosomal regions. Regulatory cis-acting elements detected in the HvSOC1-like1 promoter include the CArG-box, implicated in the regulation of SOC1 expression in Arabidopsis. Both HvSOC1-like1 and HvSOCI-like2 are expressed in vegetative and reproductive tissues and at different stages of seed development. Both are upregulated in a particular seed developmental stage suggesting their possible implication in seed development. Furthermore, HvSOC1-like1 was induced in two winter barley cultivars after vernalization treatment pointing to its probable involvement in the vernalization process. The study of the SOC1 genes reported here opens the way for a better understanding of both the vernalization process and seed development and germination in this important cereal crop.     

Molecular and dendrochronological analysis of natural root grafting in Populus tremuloides (Salicaceae)

Trembling aspen (Populus tremuloides) is a clonal tree species, which regenerates mostly through root suckering. In spite of vegetative propagation, aspen maintains high levels of clonal diversity. We hypothesized that the maintenance of clonal diversity in this species can be facilitated by integrating different clones through natural root grafts into aspen's communal root system. To verify this hypothesis, we analyzed root systems of three pure aspen stands where clones had been delineated with the help of molecular markers. Grafting between roots was frequent regardless of their genotypes. Root system excavations revealed that many roots were still living below trees that had been dead for several years. Some of these roots had no root connections other than grafts to living ramets of different clones. The uncovered root systems did not include any unique genotypes that would not occur among stems. Nevertheless, acquiring roots of dead trees helps to maintain extensive root systems, which increases the chances of clone survival. Substantial interconnectivity within clones as well as between clones via interclonal grafts results in formation of large genetically diverse physiological units. Such a clonal structure can significantly affect interpretations of diverse ecophysiological processes in forests of trembling aspen.     

Functional analysis of MADS-box genes controlling ovule development in Arabidopsis using the ethanol-inducible alc gene-expression system

In Arabidopsis, different combinations of ABC organ identity proteins interact in the presence of SEPALLATA (SEP) proteins to regulate floral organ differentiation. Ectopic expression of SEP3 in combination with class A and B or B and C genes is sufficient to homeotically convert vegetative leaves into petal-like organs and bracts into stamen-like structures, respectively. Recently, it has been shown that the three MADS-box genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHP2 act redundantly to control ovule identity. Protein interaction assays performed in yeast in combination with genetic studies demonstrated that these MADS-box factors only interact in the presence of SEP proteins to form complexes that determine ovule differentiation. Here, we address the question whether the ectopic co-expression of ovule identity proteins is sufficient to induce the homeotic conversion of vegetative leaves into carpel-like structures bearing ovules. We present the phenotypic characterization of Arabidopsis plants that ectopically express ovule identity factors under the regulation of the ethanol inducible gene expression system. These experiments indicate that the ectopic co-expression of SEP3 and SHP1 and/or STK is probably not sufficient to homeotically transform vegetative tissues into carpels with ovules. However, comparing the phenotypes obtained by ectopic expression of STK and/or SHP1 with or without SEP3 shows that co-expression of factors that are able to form complexes in yeast cause more extreme homeotic transformations, confirming the functional role of these complexes in vivo.