LEAFY Gene Research Articles

Overexpression of AtLEAFY Accelerates Flowering in Brassica juncea

The floral meristem identity gene LEAFY (LFY) from Arabidopsis thaliana (L.) Heynh. can accelerate flowering in dicotyledonous plants. In this report, we raised transgenic plants of Brassica juncea (L.) Czern. cv. Varuna carrying the LEAFY gene (LFY) in sense and antisense orientation and studied the effect of modulation of LFY expression on flowering. The time required for the initiation of flowering was found to be reduced by 19% (∼1 wk earlier) in the transgenic plants overexpressing LFY as compared with the wild‐type untransformed control plants. Moreover, no significant yield penalty was observed in these transgenic plants. Altogether, these results open up the possibility of manipulating the flowering time in B. juncea without significant yield penalty or abnormalities.

Desenvolvimento reprodutivo de Hyptis suaveolens (L.) Poit.: relação entre fotoperíodo, densidade celular meristemática e padrão de expressão de um ortólogo putativo do gene LEAFY de arabidopsis

Hyptis suaveolens (L.) Poit. (Lamiaceae) is a typical invasive plant that produces an essential oil with several biological properties. The largest essential oil content is found at the flowers. So, advances in the current knowledge about the H. suaveolens reproductive development are important when the essential oil productivity is sought. Furthermore, the comprehension of the mechanisms involved in the H. suaveolens floral induction would be useful to clarify the adaptive responses in invasive plants. We here reported some H. suaveolens reproductive development features. The plants were grown at two photoperiodic treatments (natural photoperiod from Alfenas, Minas Gerais, Brazil and natural photoperiod extended of 16 h). Samples of floral and vegetative aerial apices were taken at five different periods of time, for anatomical and scanning electron microscopic analysis, as well as in situ hybridization experiments, in order to detect the transcripts of a putative ortholog of Arabidopsis thaliana LEAFY gene. Hyptis suaveolens is a short day plant, with critical photoperiod nearby 13 h. The cell density has increased according to higher vegetative or floral meristem activities. The putative ortholog of LEAFY in H. suaveolens has shown an expression pattern similar to that reported in Antirrhinum and Arabidopsis, although it was also expressed in vegetative apices at the natural photoperiod plants. Nevertheless, under extended natural photoperiod (16 h) no gene expression was detected in vegetative meristem. These results suggest a basal expression of LEAFY in H. suaveolens vegetative meristem, which is strongly decreased by extended day length conditions, or is increased under photoperiods lower than 13 h, when suitable levels are reached for floral determination.

Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine

A genetic linkage map of grapevine was constructed using a pseudo-testcross strategy based upon 138 individuals derived from a cross of Vitis vinifera Cabernet Sauvignon x Vitis riparia Gloire de Montpellier. A total of 212 DNA markers including 199 single sequence repeats (SSRs), 11 single strand conformation polymorphisms (SSCPs) and two morphological markers were mapped onto 19 linkage groups (LG) which covered 1,249 cM with an average of 6.7 cM between markers. The position of SSR loci in the maps presented here is consistent with the genome sequence. Quantitative traits loci (QTLs) for several traits of inflorescence and flower morphology, and downy mildew resistance were investigated. Two novel QTLs for downy mildew resistance were mapped on linkage groups 9 and 12, they explain 26.0-34.4 and 28.9-31.5% of total variance, respectively. QTLs for inflorescence morphology with a large effect (14-70% of total variance explained) were detected close to the Sex locus on LG 2. The gene of the enzyme 1-aminocyclopropane-1-carboxylic acid synthase, involved in melon male organ development and located in the confidence interval of all QTLs detected on the LG 2, could be considered as a putative candidate gene for the control of sexual traits in grapevine. Co-localisations were found between four QTLs, detected on linkage groups 1, 14, 17 and 18, and the position of the floral organ development genes GIBBERELLIN INSENSITIVE1, FRUITFULL, LEAFY and AGAMOUS. Our results demonstrate that the sex determinism locus also determines both flower and inflorescence morphological traits.

Phylogeny, origin, and biogeographic history of Aesculus L. (Sapindales) – an update from combined analysis of DNA sequences, morphology, and fossils

AbstractConflicting phylogenies and biogeographic hypotheses for Aesculus L. were reevaluated using data integrating morphology, fossils, and DNA sequences. A more robust phylogeny of Aesculus was constructed by increasing taxon and character sampling to include 42 morphological characters and five DNA regions including rps16, trnHK, matK, ITS, and part of the LEAFY gene. Our analyses revealed three well‐supported major lineages in Aesculus. These are sect. Calothyrsus excluding A. californica, sect. Macrothyrsus + A. californica, and sect. Aesculus + sect. Pavia + sect. Parryana. The relationship of sect. Aesculus to sect. Parryana + sect. Pavia is strongly supported and previously unreported. Relationships among the three major clades remained incompletely resolved. Biogeographic and divergence time analyses using DIVA and Multidivtime support a Paleocene origin of Aesculus in eastern Asia and western North America, and showed that the genus subsequently dispersed into eastern North America, Central America, and Europe. Our results support that Aesculus originated as an element of the Paleogene mesophilic flora in the Beringian region with multiple eastward migrations characterizing the early spatial history of this genus. This biogeographic history supports Trans‐Beringian migration of plants in the very early Paleogene.

Nonflowers near the base of extant angiosperms? Spatiotemporal arrangement of organs in reproductive units of Hydatellaceae and its bearing on the origin of the flower

Reproductive units (RUs) of Trithuria, the sole genus of the early-divergent angiosperm family Hydatellaceae, are compared with flowers of their close relatives in Cabombaceae (Nymphaeales). Trithuria RUs combine features of flowers and inflorescences. They differ from typical flowers in possessing an "inside-out" morphology, with carpels surrounding stamens; furthermore, carpels develop centrifugally, in contrast to centripetal or simultaneous development in typical flowers. Trithuria RUs could be interpreted as pseudanthia of two or more cymose partial inflorescences enclosed within an involucre, but the bractlike involucral phyllomes do not subtend partial inflorescences and hence collectively resemble a typical perianth. Teratological forms of T. submersa indicate a tendency to fasciation and demonstrate that the inside-out structure-the primary feature that separates RUs of Hydatellaceae from more orthodox angiosperm flowers-can be at least partially modified, thus producing a morphology that is closer to an orthodox flower. The Trithuria RU could be described as a "nonflower", i.e., a structure that contains typical angiosperm carpels and stamens but does not allow recognition of a typical angiosperm flower. The term nonflower could combine cases of secondary loss of flower identity and cases of a prefloral condition, similar to those that gave rise to the angiosperm flower. Nonhomology among some angiosperm flowers could be due to iterative shifts between nonfloral construction and flower/inflorescence organization of reproductive organs. Potential testing of these hypotheses using evolutionary-developmental genetics is explored using preliminary data from immunolocalization of the floral meristem identity gene LEAFY in T. submersa, which indicated protein expression at different hierarchical levels.

Molecular markers and a sequence deletion in intron 2 of the putative partial homologue of LEAFY reveal geographical structure to genetic diversity in the acutely threatened legume genus Clianthus

Clianthus is an acutely threatened, bird-pollinated genus endemic to New Zealand, represented in the wild by only one population of C. puniceus and 11 populations of C. maximus, each with very few individuals (typically <10 per population). A limited number of named Clianthus cultivars of indeterminate origin are commonly grown as ornamentals. Genomic DNA from individual Clianthus plants was extracted for genetic diversity analysis using a range of molecular markers, including amplified fragment length polymorphism (AFLP). Data were analysed by the unweighted pair-group method with arithmetic averaging (UPGMA), the generation of Neighbor-Joining trees, and analyses of molecular variance (AMOVA). Genetic distance between wild populations of C. maximus was highly correlated with geographical distance between populations. Sequencing of intron 2 of a putative partial homologue of the floral meristem identity gene LEAFY ( CmLFY) revealed a 7 bp deletion that was exhibited homozygously in the more northern populations of C. maximus, and in all individuals tested from the sole population of C. puniceus. This deletion was not exhibited in more southern populations of C. maximus. Further, one geographically intermediate population contained some plants that were heterozygous for the deletion. Parallel analyses of cultivated Clianthus genotypes, more than half of which were also homozygous for the 7 bp deletion, showed that these were not representative of the broad, but threatened, diversity remaining in the wild. It is argued that wild populations of C. maximus are unlikely to have arisen from the escape of plants from cultivation. Conservation effort should focus on the protection and study of the extant plants in these wild populations, rather than on the introduction of disturbance regimes to uncover potential seed banks.

AGAMOUS‐LIKE 17, a novel flowering promoter, acts in a FT‐independent photoperiod pathway

The photoperiod pathway is a genetically conserved pathway that affects flowering in distantly related angiosperms. Here we report a novel flowering promoter AGAMOUS-LIKE 17 (AGL17) acting in the photoperiod pathway of Arabidopsis. AGL17 transcripts were detectable in various plant organs with the highest expression in the root. Under long-day conditions, expression of AGL17 gradually increased in the aerial part of seedlings during the floral transition. Overexpression of AGL17 caused early flowering, while loss of function of AGL17 exhibited late flowering, particularly under long days. Analysis of AGL17 expression in various flowering-time mutants showed that its transcripts were significantly reduced in the photoperiod pathway mutant co-1. Correspondingly, AGL17 expression was upregulated in transgenic plants overexpressing CONSTANS (CO) and also when CO activity was induced by light. Genetic analysis further showed that overexpression of AGL17 could partially suppress the late flowering of co-1. These results suggest that AGL17 acts to promote flowering and is positively controlled by the photoperiod pathway regulator CO. In contrast, another target of CO, FLOWERING LOCUS T (FT), did not affect AGL17 expression, and vice versa. The expression of two floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) decreased in agl17-1, while LFY and AP1 could be rapidly induced by AGL17 using a functional estradiol-inducible system. These findings indicate that AGL17 ultimately promotes flowering via regulation of LFY and AP1.

Phylogenetics of Polygonaceae with an Emphasis on the Evolution of Eriogonoideae

Abstract Polygonaceae has long been recognized as a monophyletic family but the circumscription of groups within it has been in constant flux. Two groups currently recognized are the subfamilies Eriogonoideae and Polygonoideae. An analysis using three chloroplast regions (rbcL, matK, and ndhF) and increased taxon sampling of Eriogonoideae does not support this delimitation. The second intron and portions of the flanking exons of the nuclear gene LEAFY, were used to explore phylogenetic utility in Polygonaceae. The intron showed high levels of variability and was useful at low taxonomic levels, but not alignable outside of Eriogonum and allied genera. Phylogenetic analysis of data from the flanking exons of the second intron of LEAFY generally supports the major groupings found in the chloroplast analysis. A new definition of the subfamilies is proposed. Polygonoideae is restricted to what has been recognized as tribes Persicarieae, Rumiceae, and Polygoneae, with the addition of Muehlenbeckia. Eriogonoidea...

Expression Analysis of the LFY and TFL1 Homologs in Floral Buds of Japanese Pear (Pyrus pyrifolia Nakai) and Quince (Cydonia oblonga Mill.)

We investigated the expression of Japanese pear (Pyrus pyrifolia) and quince (Cydonia oblonga) homologs of Arabidopsis thaliana flowering-related genes LEAFY (LFY) and TERMINAL FLOWER 1 (TFL1) in dissected floral tissues. Japanese pear and quince belong to the subfamily Maloideae (Rosaceae), which is an ancient polyploid; thus, the genome of each plant contains at least two different homologs of LFY and TFL1. We examined the expression of each homolog separately using gene-specific reverse transcription-polymerase chain reaction (RT-PCR). Under the experimental conditions used in this study, there was no difference in the expression pattern of LFY homologs, regardless of the species and homolog type. In contrast, TFL1 homologs were differentially expressed in Japanese pear and quince. In Japanese pear, PpTFL1-1 and PpTFL1-2 were expressed in the apical meristem prior to floral differentiation; however, during floral differentiation, PpTFL1-2 expression disappeared and PpTFL1-1 alone continued to be expressed in the developing inflorescence. In quince, CoTFL1-1 was the only TFL1 homolog detected prior to floral differentiation. Upon floral differentiation, both CoTFL1-1 and CoTFL1-2 were expressed mainly in the domed apical meristem. These expressions decreased to an undetectable level once the apical meristem had transformed into a single flower meristem. Thus, Japanese pear and quince TFL1 homologs are supposed to be involved in the formation of domed apical meristems during early reproductive development. Our results imply the importance of PpTFL1-1 in morphological development of Japanese pear inflorescence.

RsLFY, a LEAFY homologue gene in radish (Raphanus sativus), is continuously expressed in vegetative, reproductive and seed development

The floral meristem identity gene LEAFY (LFY) in Arabidopsis plays a key role in flower development. We isolated two LFY-like genes from radish, designated RsLFY1 and RsLFY2. Comparison of genomic and cDNA sequences revealed that the number and positions of introns are precisely conserved in RsLFY1, RsLFY2 and LFY. Using RsLFY1 full- length cDNA as a probe, genomic DNA blot hybridization analysis detected two hybridizing fragments under high stringency, suggesting that there are two RsLFY loci within radish genome. Both genes were expressed during vegetative growth, reproductive growth, flower development and seed development. RsLFY1 was expressed slightly in leaf primordia and strongly in early floral meristem. It was expressed successively in primordia of sepals, petals, stamens, gynoecium, ovule integument and mature seeds. These results suggest that RsLFY1 plays a similar role to LFY in floral initiation and the developmental stages of floral organs, and that it also may regulate radish seed development, unlike LFY.

Phylogenetic Analysis of Mandarin Landraces, Wild Mandarins, and Related Species in China Using Nuclear LEAFY Second Intron and Plastid trnL-trnF Sequence

Nucleotide sequences of the second intron of the nuclear LEAFY gene (FLint2) and trnL-trnF region of the chloroplast genome were used to analyze the phylogenetic relationships among eight wild mandarins (Citrus reticulata Blanco), 19 mandarin landraces, and 19 related species of Citrus L. Forty-six trnL-trnF sequences and 111 FLint2 sequences were obtained from 46 ingroup accessions, with an average length of 1059.7 and 776.7 bp respectively. Phylogeny reconstructions were conducted separately for these two data sets using maximum parsimony and maximum likelihood criteria. Monophyly of mandarins was supported by both of these data sets, and in this clade, most mandarin landraces formed an unresolved polytomy, whereas ‘Jiangyong 1’, ‘Chongyi A2’, ‘Chongyi A1’ (or ‘Jiangyong 4’ in FLint2 data), and ‘Daoxian 1’ wild mandarins formed a subclade. ‘Mangshan A1’ and ‘Daoxian 5’ wild mandarins were sisters to this mandarin clade. A hybrid origin of five mandarin landraces and several mandarin-related species was suggested as a plausible hypothesis to explain the incongruence between the FLint2 and trnL-trnF data sets.

Specification ofArabidopsisfloral meristem identity by repression of flowering time genes

Flowering plants produce floral meristems in response to intrinsic and extrinsic flowering inductive signals. In Arabidopsis, the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) are activated to play a pivotal role in specifying floral meristems during floral transition. We show here that the emerging floral meristems require AP1 to partly specify their floral identities by directly repressing a group of flowering time genes, including SHORT VEGETATIVE PHASE (SVP), AGAMOUS-LIKE 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1). In wild-type plants, these flowering time genes are normally downregulated in emerging floral meristems. In the absence of AP1, these genes are ectopically expressed, transforming floral meristems into shoot meristems. By post-translational activation of an AP1-GR fusion protein and chromatin immunoprecipitation assays, we further demonstrate the repression of these flowering time genes by induced AP1 activity and in vivo AP1 binding to the cis-regulatory regions of these genes. These findings indicate that once AP1 is activated during the floral transition, it acts partly as a master repressor in floral meristems by directly suppressing the expression of flowering time genes, thus preventing the continuation of the shoot developmental program.

Interaction of the BRACTEA gene with the TERMINAL FLOWER1, LEAFY, and APETALA1 genes during inflorescence and flower development in Arabidopsis thaliana

The major Arabidopsis thaliana genes controlling the shoot architecture are TERMINAL FLOWER1 (TFL1), APETALA1 (AP1), and LEAFY (LFY). The BRACTEA (BRA) gene also codes for one of the key regulators of inflorescence development. The bra tfl1-11, bra lfy-5, and bra ap1-20 double mutants were analyzed morphologically, and expression of the TFL1, AP1, and LFY genes was studied in the bra mutant and wild-type plants. The BRA gene was found to positively regulate the TFL1 and AP1 genes after floral initiation, determining more than 70% of their total expression level. In floral meristem, the BRA gene prevented AP1 expression, restricting AP1 transcription to the perianth zone. Since flowers were completely converted into vegetative shoots in the bra lfy-5 double mutant, it was assumed that the BRA and LFY genes function redundantly and independently in floral initiation. The results demonstrate that BRA is one of the genes that determine the balance between maintenance of proliferative activity in apical meristem and floral development in its peripheral region; such a balance is necessary for indeterminate inflorescence development.

Field trial detects incomplete barstar attenuation of vegetative cytotoxicity in Populus trees containing a poplar LEAFY promoter::barnase sterility transgene

We tested the efficacy of an attenua- tion system developed to preclude the deleteri- ous effects of floral promoter::cytotoxin genes on vegetative growth of transgenic sterile plants. We tested the promoter (2.6 kb 5¢ region) of the poplar LEAFY gene PTLF driving barstar, combined on the same T-DNA with barstar dri- ven by either the CaMV 35S basal promoter +5 to -72 fragment (35SBP), 35SBP fused to the TMV omega element (35SBP omega), or the NOS promoter. The unattenuated pPTLF::barnase construct failed to give rise to any transgenic events, suggesting substantial non-reproductive expression from this promoter. The barstar- attenuated constructs enabled transformation, but the rate was reduced by nearly one-third. Four events (7% of attenuated events) had highly abnormal morphology, and were identified during the early phases of propagation; these events had significantly higher barnase:barstar expression ra- tios based on quantitative RT-PCR. A greenhouse study showed that phenotypically normal attenu- ated plants grew at the same rate as wild-type and barnase-lacking transgenic plants. A statistically significant positive linear association was found between relative growth rate (RGR) and bar- star:barnase ratio in the attenuated events, and graphical analysis suggested a threshold for barstar attenuation of barnase, above which additional levels of barstar did not provide further attenua- tion. Surprisingly, the appearance and growth rate of the nearly all of the attenuated events were substantially reduced after one or two growing seasons in the field, and the extent of growth reduction was associated with barstar:barnase expression ratio. These results demonstrate the importance of field testing during early phases of research to identify pleiotropic effects of transgenic sterility genes in trees.

Independent origins of tetraploid cryptic species in the fern Ceratopteris thalictroides

Ceratopteris thalictroides (L.) Brongn is a tetraploid fern species that contains at least three cryptic species, the south, the north and the third type. In this study we combined data from both chloroplast DNA (cpDNA) and nuclear DNA sequences of three diploid species and three cryptic species of C. thalictroides to unravel the origin of the cryptic species, particularly of the reticulate relationships among the diploid and tetraploid taxa in the genus Ceratopteris. Of the three diploid species examined, C. cornuta had cpDNA identical to that of the tetraploid third type plants, and this diploid species is a possible maternal ancestor of the tetraploid third type. Analysis of the homologue of the Arabidopsis thaliana LEAFY gene (CLFY1) identified ten alleles in the genus Ceratopteris, with six alleles found in C. thalictroides. The unrooted tree of the CLFY1 gene revealed four clusters. Each cryptic species showed fixed heterozygosity at the CLFY1 locus and had two alleles from different clusters of the CLFY1 tree. Consideration of the cpDNA sequences, CLFY1 genotypes of the cryptic species and CLFY1 gene tree in concert suggested that the cryptic species of C. thalictroides had originated through independent allopolyploidization events involving C. cornuta and two unknown hypothetical diploid species.

GA4 Is the Active Gibberellin in the Regulation ofLEAFYTranscription andArabidopsisFloral Initiation

Flower initiation in Arabidopsis thaliana under noninductive short-day conditions is dependent on the biosynthesis of the plant hormone gibberellin (GA). This dependency can be explained, at least partly, by GA regulation of the flower meristem identity gene LEAFY (LFY) and the flowering time gene SUPPRESSOR OF CONSTANS1. Although it is well established that GA(4) is the active GA in the regulation of Arabidopsis shoot elongation, the identity of the GA responsible for the regulation of Arabidopsis flowering has not been established. Through a combination of GA quantifications and sensitivity assays, we show that GA(4) is the active GA in the regulation of LFY transcription and Arabidopsis flowering time under short-day conditions. The levels of GA(4) and sucrose increase dramatically in the shoot apex shortly before floral initiation, and the regulation of genes involved in GA metabolism suggests that this increase is possibly due to transport of GAs and sucrose from outside sources to the shoot apex. Our results demonstrate that in the dicot Arabidopsis, in contrast with the monocot Lolium temulentum, GA(4) is the active GA in the regulation of both shoot elongation and flower initiation.

Dating Dispersal and Radiation in the Gymnosperm Gnetum (Gnetales)—Clock Calibration When Outgroup Relationships Are Uncertain

Most implementations of molecular clocks require resolved topologies. However, one of the Bayesian relaxed clock approaches accepts input topologies that include polytomies. We explored the effects of resolved and polytomous input topologies in a rate-heterogeneous sequence data set for Gnetum, a member of the seed plant lineage Gnetales. Gnetum has 10 species in South America, 1 in tropical West Africa, and 20 to 25 in tropical Asia, and explanations for the ages of these disjunctions involve long-distance dispersal and/or the breakup of Gondwana. To resolve relationships within Gnetum, we sequenced most of its species for six loci from the chloroplast (rbcL, matK, and the trnT-trnF region), the nucleus (rITS/5.8S and the LEAFY gene second intron), and the mitochondrion (nad1 gene second intron). Because Gnetum has no fossil record, we relied on fossils from other Gnetales and from the seed plant lineages conifers, Ginkgo, cycads, and angiosperms to constrain a molecular clock and obtain absolute times for within-Gnetum divergence events. Relationships among Gnetales and the other seed plant lineages are still unresolved, and we therefore used differently resolved topologies, including one that contained a basal polytomy among gymnosperms. For a small set of Gnetales exemplars (n = 13) in which rbcL and matK satisfied the clock assumption, we also obtained time estimates from a strict clock, calibrated with one outgroup fossil. The changing hierarchical relationships among seed plants (and accordingly changing placements of distant fossils) resulted in small changes of within-Gnetum estimates because topologically closest constraints overrode more distant constraints. Regardless of the seed plant topology assumed, relaxed clock estimates suggest that the extant clades of Gnetum began diverging from each other during the Upper Oligocene. Strict clock estimates imply a mid-Miocene divergence. These estimates, together with the phylogeny for Gnetum from the six combined data sets, imply that the single African species of Gnetum is not a remnant of a once Gondwanan distribution. Miocene and Pliocene range expansions are inferred for the Asian subclades of Gnetum, which stem from an ancestor that arrived from Africa. These findings fit with seed dispersal by water in several species of Gnetum, morphological similarities among apparently young species, and incomplete concerted evolution in the nuclear ITS region.

Transgenic sterility in Populus: expression properties of the poplar PTLF, Agrobacterium NOS and two minimal 35S promoters in vegetative tissues

Transgenic sterility is a desirable trait for containment of many kinds of transgenes and exotic species. Genetically engineered floral sterility can be imparted by expression of a cytotoxin under the control of a predominantly floral-tissue-specific promoter. However, many otherwise desirable floral promoters impart substantial non-floral expression, which can impair plant health or make it impossible to regenerate transgenic plants. We are therefore developing a floral sterility system that is capable of attenuating undesired background vegetative expression. As a first step towards this goal, we compared the vegetative expression properties of the promoter of the poplar (Populus trichocarpa Torr. & Gray) homolog of the floral homeotic gene LEAFY (PTLF), which could be used to impart male and female flower sterility, to that of three candidate attenuator-gene promoters: the cauliflower mosaic virus (CaMV) 35S basal promoter, the CaMV 35S basal promoter fused to the TMV omega element and the nopaline synthase (NOS) promoter. The promoters were evaluated via promoter::GUS gene fusions in a transgenic poplar hybrid (Populus tremula L. x P. alba L.) by both histochemical and fluorometric GUS assays. In leaves, the NOS promoter conveyed the highest activity and had a mean expression level 5-fold higher than PTLF, whereas the CaMV 35S basal promoter fused to the omega element and the CaMV 35S basal promoter alone directed mean expression levels that were 0.5x and 0.35x that of PTLF, respectively. Differential expression in shoots, leaves, stems and roots was observed only for the NOS and PTLF promoters. Strongest expression was observed in roots for the NOS promoter, whereas the PTLF promoter directed highest expression in shoots. The NOS promoter appears best suited to counteract vegetative expression of a cytotoxin driven by the PTLF promoter where 1:1 toxin:attenuator expression is required.

Genetics of Resistance to Field Kernel Infection by Aspergillus flavus in Maize

To solve the chronic problem of maize (Zea mays L.) kernel infection by Aspergillus flavus in the southeastern USA, genetic resistance to A. flavus is needed. A 2-yr study of a diallel cross of five parents containing the leafy gene (Lfy) was conducted to evaluate the inhertance of resistance to A. flavus during 2003 and 2004. The maize percent kernel infection (PKI) rates were determined via the media-free and kernel-isolated incubation method. Highly significant general combining ability (GCA), specific combining ability (SCA), and reciprocal effects were found. Parents 914 and A619 had desirable (negative) GCA effects to enhance the average performance of A. flavus resistance in hybrid progeny. The crosses 914 X A632, 914 X Wf9, and Hy X Wf9 had consistently negative SCA effects across years. The reciprocal effects were detected, indicating the presence of maternal effects in the maize kernel and the importance of direction of a cross. For example, A632 X Hy had the largest positive significant reciprocal effect; the reversal of this cross, i.e., Hy X A632, should impart resistance to progeny. A North Carolina Design-II analysis of four female lines (914, A632, HY, and Wf9) crossed with three male lines (A619, B73, and Mol7), was used to estimate additive and dominance genetic variances. Narrow-sense heritability for PKI was 37.6% and broad-sense heritability estimate was 89.1%. Breeding procedures to handle both additive and dominance variances would need to be used to improve resistance to PKI.

Polymorphism of the LEAFY Gene in Brassica Plants

The arabidopsis gene LEAFY controls the induction of flowering and maintenance of the floral meristem identity. By comparing the primary structure of LEAFY and its homologs in other Brassicaceae species and beyond this family, we singled out four clusters corresponding to three systematically remote families of angiosperms, Brassicaceae, Solanaceae, and Poaceae, and to gymnosperms. Both structural and functional distinctions of LEAFY homologs from their arabidopsis prototype expanded in the range Brassicaceae—Solanaceae—Poaceae. A LEAFY homolog from B. juncea cloned in our laboratory was used as a hybridization probe to analyze the restriction fragment length polymorphism in six Brassica species comprising diploid (AA, BB, and CC) and allotetraploid (AABB, AACC, and BBCC) genomes. In this way we recognized LEAFY fragments specific of genomes A, B, and C; in contrast, the variations of the length and structure of the LEAFY intron 2 were not genome-specific. LEAFY polymorphism in the Brassica accessions comprising genome B was related to their geographic origin and apparently to the adaptation to day length.