The WUSCHEL-RELATED HOMEOBOX 3 gene PaWOX3 regulates lateral organ formation in Norway spruce.

In angiosperms, WUSCHEL-RELATED HOMEOBOX 3 (WOX3) genes are required for the recruitment of founder cells from the lateral domains of shoot meristems that form lateral regions of leaves. However, the regulation of the formation of lateral organs in gymnosperms remains unknown. By using somatic embryos of Norway spruce (Picea abies) we have studied the expression and function of PaWOX3 during embryo development. The mRNA abundance of PaWOX3 was determined by quantitative real-time PCR, and the spatial expression of PaWOX3 was analysed by histochemical ß-glucuronidase (GUS) assays and in situ mRNA hybridization. To investigate the function of PaWOX3, we analysed how downregulation of PaWOX3 in RNA interference lines affected embryo development and morphology. PaWOX3 was highly expressed in mature embryos at the base of each cotyledon close to the junction between the cotyledons, and in the lateral margins of cotyledons and needles, separating them into an adaxial and an abaxial side. Downregulation of the expression of PaWOX3 caused defects in lateral margin outgrowth in cotyledons and needles, and reduced root elongation. Our data suggest that the WOX3 function in margin outgrowth in lateral organs is conserved among the seed plants, whereas its function in root elongation may be unique to gymnosperms.

Molecular control of normal and acrocona mutant seed cone development in Norway spruce (Picea abies) and the evolution of conifer ovule-bearing organs.

Reproductive organs in seed plants are morphologically divergent and their evolutionary history is often unclear. The mechanisms controlling their development have been extensively studied in angiosperms but are poorly understood in conifers and other gymnosperms. Here, we address the molecular control of seed cone development in Norway spruce, Picea abies. We present expression analyses of five novel MADS-box genes in comparison with previously identified MADS and LEAFY genes at distinct developmental stages. In addition, we have characterized the homeotic transformation from vegetative shoot to female cone and associated changes in regulatory gene expression patterns occurring in the acrocona mutant. The analyses identified genes active at the onset of ovuliferous and ovule development and identified expression patterns marking distinct domains of the ovuliferous scale. The reproductive transformation in acrocona involves the activation of all tested genes normally active in early cone development, except for an AGAMOUS-LIKE6/SEPALLATA (AGL6/SEP) homologue. This absence may be functionally associated with the nondeterminate development of the acrocona ovule-bearing scales. Our morphological and gene expression analyses give support to the hypothesis that the modern cone is a complex structure, and the ovuliferous scale the result of reductions and compactions of an ovule-bearing axillary short shoot in cones of Paleozoic conifers.

Morphological "primary homology" and expression of AG-subfamily MADS-box genes in pines, podocarps, and yews.

The morphological variation among reproductive organs of extant gymnosperms is remarkable, especially among conifers. Several hypotheses concerning morphological homology between various conifer reproductive organs have been put forward, in particular in relation to the pine ovuliferous scale. Here, we use the expression patterns of orthologs of the ABC-model MADS-box gene AGAMOUS (AG) for testing morphological homology hypotheses related to organs of the conifer female cone. To this end, we first developed a tailored 3'RACE procedure that allows reliable amplification of partial sequences highly similar to gymnosperm-derived members of the AG-subfamily of MADS-box genes. Expression patterns of two novel conifer AG orthologs cloned with this procedure-namely PodAG and TgAG, obtained from the podocarp Podocarpus reichei and the yew Taxus globosa, respectively-are then further characterized in the morphologically divergent female cones of these species. The expression patterns of PodAG and TgAG are compared with those of DAL2, a previously discovered Picea abies (Pinaceae) AG ortholog. By treating the expression patterns of DAL2, PodAG, and TgAG as character states mapped onto currently accepted cladogram topologies, we suggest that the epimatium-that is, the podocarp female cone organ previously postulated as a "modified" ovuliferous scale-and the canonical Pinaceae ovuliferous scale can be legitimally conceptualized as "primary homologs." Character state mapping for TgAG suggests in turn that the aril of Taxaceae should be considered as a different type of organ. This work demonstrates how the interaction between developmental-genetic data and formal cladistic theory could fruitfully contribute to gymnosperm systematics.

B-function expression in the flower center underlies the homeotic phenotype of Lacandonia schismatica (Triuridaceae).

Spontaneous homeotic transformations have been described in natural populations of both plants and animals, but little is known about the molecular-genetic mechanisms underlying these processes in plants. In the ABC model of floral organ identity in Arabidopsis thaliana, the B- and C-functions are necessary for stamen morphogenesis, and C alone is required for carpel identity. We provide ABC model-based molecular-genetic evidence that explains the unique inside-out homeotic floral organ arrangement of the monocotyledonous mycoheterotroph species Lacandonia schismatica (Triuridaceae) from Mexico. Whereas a quarter million flowering plant species bear central carpels surrounded by stamens, L. schismatica stamens occur in the center of the flower and are surrounded by carpels. The simplest explanation for this is that the B-function is displaced toward the flower center. Our analyses of the spatio-temporal pattern of B- and C-function gene expression are consistent with this hypothesis. The hypothesis is further supported by conservation between the B-function genes of L. schismatica and Arabidopsis, as the former are able to rescue stamens in Arabidopsis transgenic complementation lines, and Ls-AP3 and Ls-PI are able to interact with each other and with the corresponding Arabidopsis B-function proteins in yeast. Thus, relatively simple molecular modifications may underlie important morphological shifts in natural populations of extant plant taxa.

The MADS-box gene DAL1 is a potential mediator of the juvenile-to-adult transition in Norway spruce (Picea abies).

Progression through the plant life cycle involves change in many essential features, most notably in the capacity to reproduce. The transition from a juvenile vegetative and non-reproductive to an adult reproductive phase is gradual and can take many years; in the conifer Norway spruce, Picea abies, typically 20-25 years. We present a detailed analysis of the activities of three regulatory genes with potential roles in this transition in Norway spruce: DAL1, a MADS-box gene related to the AGL6 group of genes from angiosperms, and the two LEAFY-related genes PaLFY and PaNLY. DAL1 activity is initiated in the shoots of juvenile trees at an age of 3-5 years, and then increases with age, whereas both LFY genes are active throughout the juvenile phase. The activity of DAL1 further shows a spatial pattern along the stem of the tree that parallels a similar gradient in physiological and morphological features associated with maturation to the adult phase. Constitutive expression of DAL1 in transgenic Arabidopsis plants caused a dramatic attenuation of both juvenile and adult growth phases; flowers forming immediately after the embryonic phase of development in severely affected plants. Taken together, our results support the notion that DAL1 may have a regulatory role in the juvenile-to-adult transition in Norway spruce.

High-throughput screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis and mass spectrometry.

A high-throughput pKa screening method based on pressure-assisted capillary electrophoresis (CE) and mass spectrometry (MS) is presented. Effects of buffer type and ionic strength on sensitivity and pKa values were investigated. Influence of dimethyl sulfoxide (DMSO) concentration present in the sample on effective mobility measurement was examined. A series of ten volatile buffers, covering a pH range from 2.5 to 10.5 with the same ionic strength, was employed. The application of volatile background electrolytes resulted in significant signal increase as compared with commonly used non-volatile phosphate buffers. In general, the CE/MS system provided a ten-fold higher sensitivity than conventional UV detection. The newly developed CE/MS method offers high-throughput capacity by pooling a number of compounds into a single sample. Simultaneous measurement of more than 50 compounds was readily achieved in less than 150 min. The measured pKa values are consistent with the published data obtained from the CE/UV method and are also in good agreement with data generated by other methods. Other advantages of using CE/MS for pKa screening are illustrated with typical examples, including poorly soluble compounds and non-UV-absorbing compounds.

The DAL10 gene from Norway spruce (Picea abies) belongs to a potentially gymnosperm-specific subclass of MADS-box genes and is specifically active in seed cones and pollen cones.

Transcription factors encoded by different members of the MADS-box gene family have evolved central roles in the regulation of reproductive organ development in the flowering plants, the angiosperms. Development of the stamens and carpels, the pollen- and seed-bearing organs, involves the B- and C-organ-identity MADS-box genes. B- and C-type gene orthologs with activities specifically in developing pollen- and seed-bearing organs are also present in the distantly related gymnosperms: the conifers and the gnetophytes. We now report on the characterization of DAL10, a novel MADS-box gene from the conifer Norway spruce, which unlike the B- and C-type conifer genes shows no distinct orthology relationship to any angiosperm gene or clade in phylogenetic analyses. Like the B- and C-type genes, it is active specifically in developing pollen cones and seed cones. In situ RNA localization experiments show DAL10 to be expressed in the cone axis, which carry the microsporophylls of the young pollen cone. In contrast, in the seed cone it is expressed both in the cone axis and in the bracts, which subtend the ovuliferous scales. Expression data and the phenotype of transgenic Arabidopsis plants expressing DAL10 suggest that the gene may act upstream to or in concert with the B- and C-type genes in the establishment of reproductive identity of developing cones.