Activation of the <Emphasis Type="Italic">WUS</Emphasis> gene induces ectopic initiation of floral meristems on mature stem surface in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A gain-of-function Arabidopsis mutant was identified via activation tagging genetic screening. The mutant exhibited clustered ectopic floral buds on the surface of inflorescence stems. The mutant was designated as sef for stem ectopic flowers. Our detailed studies indicate that the ectopic flower meristems are initiated from the differentiated cortex cells. Inverse PCR and sequence analysis indicated that the enhancer-containing T-DNA from the activation tagging construct, SKI015, was inserted upstream of the previously cloned WUS gene encoding a homeodomain protein. Studies from RT-PCR, RNA in situ hybridization and transgenic plant analysis further confirmed that the phenotypes of sef are caused by the overexpression of WUS. Our results suggest that overexpression of WUS could trigger the cell pluripotence and reestablish a new meristem in cortex. The type of new meristems caused by WUS overexpression was dependent upon the developmental and physiological stages of a plant. With the help of some undefined factors in the reproductive organs the new meristems differentiated into floral buds. In a vegetative growth plant, however, only the new vegetative buds can be initiated upon the overexpression of WUS. These studies provide new insights of WUS on flower development.     

A “Nonsterile” Method for Selecting and Growing <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Transformants (T<Subscript>2</Subscript> Transgenic Lines) Resistant to Kanamycin

A breakthrough in transgenic Arabidopsis thaliana research was the development of the floral dip transformation protocol, a simple and reliable method of obtaining transformants, T₁ transgenic lines, at high efficiency while avoiding the use of tissue culture. However, the traditional protocol (a “sterile” method) of obtaining T₂ transgenic lines has not evolved along with improvements in transformation technology as it continues to be laborious and time-consuming. In this study, we report on the development of an improved protocol (a “nonsterile” method) for selecting and growing A. thaliana transformants (T₂ transgenic lines) resistant to kanamycin under nonsterile conditions. This protocol involves the use of a simple yet specialized device that will aid in solium selection of T₂ transgenic lines that can be rapidly grown in a hydroponic system. The “nonsterile” method reduces labor and time involved as compared to the “sterile” method; moreover, it is easy to set up and maintain. This method may also be applicable to other selecting agents, and perhaps to other plants.     

Characterization of candidate class A,B and E floral homeotic genes from the perianthless basal angiosperm <Emphasis Type="Italic">Chloranthus spicatus</Emphasis> (Chloranthaceae)

The classic ABC model explains the activities of each class of floral homeotic genes in specifying the identity of floral organs. Thus, changes in these genes may underlay the origin of floral diversity during evolution. In this study, three MADS-box genes were isolated from the perianthless basal angiosperm Chloranthus spicatus. Sequence and phylogenetic analyses revealed that they are AP1-like, AP3-like and SEP3-like genes, and hence these genes were termed CsAP1, CsAP3 and CsSEP3, respectively. Due to these assignments, they represent candidate class A, class B and class E genes, respectively. Expression patterns suggest that the CsAP1, CsAP3 and CsSEP3 genes function during flower development of C. spicatus. CsAP1 is expressed broadly in the flower, which may reflect the ancestral function of SQUA-like genes in the specification of inflorescence and floral meristems rather than in patterning of the flower. CsAP3 is exclusively expressed in male floral organs, providing the evidence that AP3-like genes have ancestral function in differentiation between male and female reproductive organs. CsSEP3 expression is not detectable in spike meristems, but its mRNA accumulates throughout the flower, supporting the view that SEP-like genes have conserved expression pattern and function throughout angiosperm. Studies of synonymous vs nonsynonymous nucleotide substitutions indicate that these genes have not evolved under changes in evolutionary forces. All the data above suggest that the genes may have maintained at least some ancestral functions despite the lack of perianth in the flowers of C. spicatus. Nucleotide sequences data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers AY316311, AY397762 and AY379963.     

对增强辣椒疫霉菌抗性的研究

病原物诱导型启动子能精确控制抗病基因在侵染位点的表达,是抗病基因工程的有效工具。prp1-1是来自马铃薯谷胱甘肽巯基转移酶基因启动子的一个273bp的片段,能够快速准确地启动被侵染位点抗病基因的表达;Rs-AFP2是具有对致病性丝状真菌的广谱抗性。该研究构建prp1-1调控Rs-AFP2基因表达的载体,经农杆菌介导转化法导入辣椒。逆转录PCR检测发现,转基因辣椒只在受到疫霉菌孢子侵染时,才由prp1-1启动Rs-AFP2基因的转录。用疫霉菌孢子灌根接种转基因辣椒T1代植株,35株T1代辣椒中有29株表现出明显的疫霉菌抗性。另将23株T1代辣椒种于人工气候箱,发现其形态和发育特征与相同条件下的非转基因植株无明显区别。研究表明,prp1-1调控Rs-AFP2的诱导表达达到了增强辣椒疫霉菌抗性的目的,而且避免了负面效应的发生。     

The Production of Marker-Free Genetically Engineered Broccoli with Sense and Antisense ACC synthase 1 and ACC oxidases 1 and 2 to Extend Shelf-Life

The production of transgenic broccoli (Brassica oleracea) with increased shelf-life using an Agrobacterium rhizogenes-mediated co-transformation protocol is reported. An Agrobacterium rhizogenes Ri vector, pRi1855:GFP was constructed to allow expression of the green fluorescent protein to identify insertion of Ri T_L-DNA into plant cells. The Brassica oleracea ACC synthase 1 and ACC oxidase 1 and 2 cDNAs in sense and antisense orientations were co-transformed into GDDH33, a doubled haploid calabrese-broccoli cultivar. Transformation efficiency was 3.26%, producing 150 transgenic root lines, of which 18 were regenerated into mature plants. The floral buds from T₀ broccoli heads were assayed for post-harvest production of ethylene and chlorophyll levels. Buds from T₀ lines transformed with ACC oxidase 1 and 2 constructs produced significantly less post-harvest ethylene at 20 °C than the untransformed plants and chlorophyll loss was significantly reduced over a 96 h post-harvest period. The T₀ plants transformed with sense and antisense ACC synthase 1 had a significantly reduced 24 h post-harvest ethylene peak and delayed chlorophyll loss. A positive correlation between post-harvest bud ethylene production and chlorophyll loss was described by a regression. This demonstrates that the shelf-life of a very perishable vegetable may be increased up to 2 days at 20 °C by reducing post-harvest ethylene production.     

Efficient transformation of beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis>) and production of plants with improved salt-tolerance

Small bud tips of 1–3 mm in length were taken from multiple shoot clumps that derived from immature inflorescence cultures of beet as recipient for the Agrobacterium-mediated transformation and transgenic plants were obtained from eight genotypes. The optimal genetic transformation protocol was established as followed: the buds were immersed in Agrobacterium suspension of OD₆₀₀ =0.3–0.5 for 5–10 min, with vacuum infiltration (0.3–0.5 × 10⁵ Pa) or supplemented with 0.01% Silwet L-77, co-cultured for 2–4 days and followed by 10-day culture on medium containing 100 mg l⁻¹ cefotaxime, then the buds were selected on medium containing 10 mg l⁻¹ hygromycin B for three consecutive generations. The percentage of hygromycin-resistant buds after three selections varied from 13.3 to 30.6% with genotypes. The results of PCR and further Southern blotting of genomic DNA of hygromycin-resistant buds or plants showed that the exogenous hpt and AtNHX1 gene had been integrated into the genomes of some transformed buds or plants. The transgenic buds or plants with AtNHX1 gene encoding Na⁺/H⁺ antiport on the vacuole membrane of Arabidopsis showed improved salt-tolerance than the controls. AtNHX1gene inherited in some transgenic lines as Mendelian segregation. This result revealed that it was feasible to improve salt-tolerance of beets by the introduction of AtNHX1 gene into cultured buds.     

Ectopic Expression of an <Emphasis Type="Italic">FT</Emphasis> Homolog from <Emphasis Type="Italic">Citrus</Emphasis> Confers an Early Flowering Phenotype on Trifoliate Orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.)

Citrus FT (CiFT) cDNA, which promoted the transition from the vegetative to the reproductive phase in Arabidopsis thaliana, when constitutively expressed was introduced into trifoliate orange (Poncirus trifoliata L. Raf.). The transgenic plants in which CiFT was expressed constitutively showed early flowering, fruiting, and characteristic morphological changes. They started to flower as early as 12 weeks after transfer to a greenhouse, whereas wild-type plants usually have a long juvenile period of several years. Most of the transgenic flowers developed on leafy inflorescences, apparently in place of thorns; however, wild-type adult trifoliate orange usually develops solitary flowers in the axils of leaves. All of the transgenic lines accumulated CiFT mRNA in their shoots, but there were variations in the accumulation level. The transgenic lines showed variation in phenotypes, such as time to first flowering and tree shape. In F₁ progeny obtained by crossing ‘Kiyomi’ tangor (C. unshiu × sinensis) with the pollen of one transgenic line, extremely early flowering immediately after germination was observed. The transgene segregated in F₁ progeny in a Mendelian fashion, with complete co-segregation of the transgene and the early flowering phenotype. These results showed that constitutive expression of CiFT can reduce the generation time in trifoliate orange.     

UFO in the Arabidopsis inflorescence apex is required for floral-meristem identity and bract suppression

The UNUSUAL FLORAL ORGANS (UFO) gene of Arabidopsis encodes an F-box protein required for the determination of floral-organ and floral-meristem identity. Mutation of UFO leads to dramatic changes in floral-organ type which are well-characterized whereas inflorescence defects are more subtle and less understood. These defects include an increase in the number of secondary inflorescences, nodes that alternate between forming flowers and secondary inflorescences, and nodes in which a single flower is subtended by a bract. Here, we show how inflorescence defects correlate with the abnormal development of floral primordia and establish a temporal requirement for UFO in this process. At the inflorescence apex of ufo mutants, newly formed primordia are initially bract-like. Expression of the floral-meristem identity genes LFY and AP1 are confined to a relatively small adaxial region of these primordia with expression of the bract-identity marker FIL observed in cells that comprise the balance of the primordia. Proliferation of cells in the adaxial region of these early primordia is delayed by several nodes such that primordia appear “chimeric” at several nodes, having visible floral and bract components. However, by late stage 2 of floral development, growth of the bract generally ceases and is overtaken by development of the floral primordium. This abnormal pattern of floral meristem development is not rescued by expression of UFO from the AP1 promoter, indicating that UFO is required prior to AP1 activation for normal development of floral primordia. We propose that UFO and LFY are jointly required in the inflorescence meristem to both promote floral meristem development and inhibit, in a non-cell autonomous manner, growth of the bract.Shelley R. Hepworth and Jennifer E. Klenz contributed equally to this work.     

Expression of a Novel Antiporter Gene from Brassica napus Resulted in Enhanced Salt Tolerance in Transgenic Tobacco Plants

Tobacco leaf discs were transformed with a plasmid pBIBnNHX1, containing the selectable marker neomycin phosphotransferase gene (nptII) and Na⁺/H⁺ vacuolar antiporter gene from Brassica napus (BnNHX1), via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the BnNHX1 gene had integrated into plant genome and Northern blot analysis revealed the transgene expression at various levels in transgenic plants. Transgenic plants expressing BnNHX1 had enhanced salt tolerance and could grow and produce seeds normally in the presence of 200 mM NaCl. Analysis for the T₁ progenies derived from seven independent transgenic primary transformants expressing BnNHX1 showed that the transgenes in most tested independent T₁ lines were inherited at Mendelian 3:1 segregation ratios. Transgenic T₁ progenies could express _BnNHX1 and had salt tolerance at levels comparable to their T₀ parental lines. This study implicates that the BnNHX1 gene represents a promising candidate in the development of crops for enhanced salt tolerance by genetic engineering.     

CENTRORADIALIS/TERMINAL FLOWER 1 gene homolog is conserved inN. tabacum, a determinate inflorescence plant

A mutation in theCENTRORADIALIS (CEN) gene ofAntirrhinum and in theTERMINAL FLOWER 1 (TFL1) gene ofArabidopsis causes their indeterminate inflorescence to determinate. We clonedCEN/TFL1 homologs fromNicotiana tabacum, the wild-type of which has a determinate inflorescence. TheCEN gene was expressed in the inflorescnece meristem and kept its inflorescence meristem identity, whereas the tobacco homolog (NCH) was expressed at a low level throughout the plant’s development. AlthoughCEN andNCH are highly homologous genes, they may have been recruited to different developmental functions during their evolution. TwoNCH genes are derived from amphidiploidN. tabacum, but both of them hybridized with its diploid parents,N. sylvestris andN. tomentosiformis. Southern blotting, and the genomic organization ofTFL1 inArabidopsis revealed that anotherCEN homolog exists in the genome ofArabidopsis. These results suggest that there are two copies of theCEN homolog per diploid plant. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology” These two authors contributed to this work equally.     

Expression of ipt gene controlled by an ethylene and auxin responsive fragment of the LEACO1 promoter increases flower number in transgenic Nicotiana tabacum

Cytokinins play important roles in regulating plant growth and development. A new genetic construct for regulating cytokinin content in plant cells was cloned and tested. The gene coding for isopentenyl transferase (ipt) was placed under the control of a 0.821 kb fragment of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene promoter from Lycopersicon esculentum (LEACO1) and introduced into Nicotiana tabacum (cv. Havana). Some LEACO1_{0.821 kb}-ipt transgenic plant lines displayed normal shoot morphology but with a dramatic increase in the number of flower buds compared to nontransgenic plants. Other transgenic lines produced excessive lateral branch development but no change in flower bud number. Isolated leaves of transgenic tobacco plants showed a significantly prolonged retention of chlorophyll under dark incubation (25°C for 20 days). Leaves of nontransformed plants senesced gradually under the same conditions. Experiments with LEACO1_{0.821 kb}-gus transgenic tobacco plants suggested auxin and ethylene involvement in induction of LEACO1_{0.821 kb} promoter activity. Multiple copies of nucleotide base sequences associated with either ethylene or auxin response elements were identified in the LEACO1_{0.821 kb} promoter fragment. The LEACO1_{0.821 kb}-ipt fusion gene appears to have potential utility for improving certain ornamental and agricultural crop species by increasing flower bud initiation and altering branching habit.     

Determination for inflorescence development is a stable state,separable from determination for flower development in Pisum sativum L. buds

Terminal meristems of Pisum sativum (garden pea) transit from vegetative to inflorescence development, and begin producing floral axillary meristems. Determination for inflorescence development was assessed by culturing excised buds and meristems. The first node of floral initiation (NFI) for bud expiants developing in culture and for adventitious shoots forming on cultured meristems was compared with the NFI of intact control buds. When terminal buds having eight leaf primordia were excised from plants of different ages (i.e., number of unfolded leaves) and cultured on 6-benzylaminopurine and kinetin-supplemented medium, the NFI was a function of the age of the source plant. By age 3, all terminal buds were determined for inflorescence development. Determination occurred at least eight nodes before the first axillary flower was initiated. Thus, the axillary meristems contributing to the inflorescence had not formed at the time the bud was explanted. Similar results were obtained for cultured axillary buds. In addition, meristems excised without leaf primordia from axillary buds three nodes above the cotyledons of age-3 plants gave rise to adventitious buds with an NFI of 8.3 ±0.3 nodes. In contrast seed-derived plants had an NFI of 16.5 ±0.2. Thus cells within the meristem were determined for inflorescence development. These findings indicate that determination for inflorescence development in P. sativum is a stable developmental state, separable from determination for flower development, and occurring prior to initiation of the inflorescence at the level of meristems.     

The tropical cedar tree (Cedrela fissilis Vell., Meliaceae) homolog of the Arabidopsis LEAFY gene is expressed in reproductive tissues and can complement Arabidopsis leafy mutants

A homolog of FLORICAULA/LEAFY, CfLFY (for Cedrela fissilis LFY), was isolated from tropical cedar. The main stages of the reproductive development in C. fissilis were documented by scanning electron microscopy and the expression patterns of CfLFY were studied during the differentiation of the floral meristems. Furthermore, the biological role of the CfLFY gene was assessed using transgenic Arabidopsis plants. CfLFY showed a high degree of similarity to other plant homologs of FLO/LFY. Southern analysis showed that CfLFY is a single-copy gene in the tropical cedar genome. Northern blot analysis and in situ hybridization results showed that CfLFY was expressed in the reproductive buds during the transition from vegetative to reproductive growth, as well as in floral meristems and floral organs but was excluded from the vegetative apex and leaves. Transgenic Arabidopsis lfy26 mutant lines expressing the CfLFY coding region, under the control of the LFY promoter, showed restored wild-type phenotype. Taken together, our results suggest that CfLFY is a FLO/LFY homolog probably involved in the control of tropical cedar reproductive development. Accession numbers: AY633621 (CfLFY gene) and AY633622 (CfLFY mRNA)     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Arabidopsis inflorescence architecture requires the activities of KNOX-BELL homeodomain heterodimers

In flowering plants, post-embryonic development is mediated by the activity of shoot and root apical meristems. Shoot architecture results from activity of the shoot apical meristem (SAM), which initiates primordia, including leaves, internodes and axillary meristems, repetitively from its flanks. Axillary meristems can develop into secondary shoots or flowers. In Arabidopsis, two paralogous BEL1-like (BELL) homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), expressed in the SAM, encode DNA-binding proteins that are essential for specifying floral primordia and establishing early internode patterning events during inflorescence development. Biochemical studies show that PNY associates with the knotted1-like homeobox (KNOX) proteins, SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP). PNY-BP heterodimers are essential for establishing early internode patterning events, while PNY-STM heterodimers are critical for SAM function. In this report, we examined the role of PNY, PNF and STM during development. First, we show that PNF interacts with STM and BP indicating that PNY and PNF are redundant functioning proteins. Inflorescence development, but not vegetative development, is sensitive to the dosage levels of PNY, PNF and STM. Characterization of stm-10, a weak allele in the Columbia ecotype, indicates that STM is also involved in floral specification and internode development. Our examination of the genetic requirements for PNY, PNF and STM demonstrates that these KNOX–BELL heterodimers control floral specification, internode patterning and the maintenance of boundaries between initiating floral primordia and the inflorescence meristem.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .