Pharbitis class-1 knotted-like homeobox gene, PKn3, shares similar characteristics to those of class-2 knotted-like genes

Three novel homeobox genes, PKn1–3 (Pharbitis knotted-like), were isolated from Japanese morning glory (Pharbitis nil Chois, strain Violet). A sequence analysis showed that these genes belong to the knotted class-1 gene family and that PKn3 has a relatively unique sequence. All PKn genes are expressed in shoot apices, stems and roots, but not in cotyledons. Transformed tobacco with PKn1 or PKn2 displayed leaf shrinkage and a dwarf phenotype, while the ectopic expression of PKn3 gave no altered phenotypes. In situ hybridization showed that PKn3 is up-regulated in developing leaf primordia and that this expression becomes restricted in the basal region of young leaf blades, which is reminiscent of the expression pattern of the class-2 knotted gene, NTH23. These data suggest that these Pharbitis homeobox genes participate in the differentiation in shoots and suggest a unique function of PKn3 in developing leaves. Received: 16 October 1999 / Revision received: 30 November 1999 / Accepted: 11 January 2000     

Suppression of a vegetative MADS box gene of potato activates axillary meristem development

Potato MADS box 1 (POTM1) is a member of the SQUAMOSA-like family of plant MADS box genes isolated from an early stage tuber cDNA library. The RNA of POTM1 is most abundant in vegetative meristems of potato (Solanum tuberosum), accumulating specifically in the tunica and corpus layers of the meristem, the procambium, the lamina of new leaves, and newly formed axillary meristems. Transgenic lines with reduced levels of POTM1 mRNA exhibited decreased apical dominance accompanied by a compact growth habit and a reduction in leaf size. Suppression lines produced truncated shoot clusters from stem buds and, in a model system, exhibited enhanced axillary bud growth instead of producing a tuber. This enhanced axillary bud growth was not the result of increased axillary bud formation. Tuber yields were reduced and rooting of cuttings was strongly inhibited in POTM1 suppression lines. Both starch accumulation and the activation of cell division occurred in specific regions of the vegetative meristems of the POTM1 transgenic lines. Cytokinin levels in axillary buds of a transgenic suppression line increased 2- to 3-fold. These results imply that POTM1 mediates the control of axillary bud development by regulating cell growth in vegetative meristems.     

Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic Arabidopsis plants

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development.     

Over-expression of a tobacco homeobox gene, NTH15 , decreases the expression of a gibberellin biosynthetic gene encoding GA 20-oxidase

Ectopic expression of the homeobox gene, NTH15 ( Nicotiana tabacum homeobox 15) in transgenic tobacco leads to abnormal leaf and flower morphology, accompanied by a decrease in the content of the active gibberellin, GA₁. Quantitative analysis of intermediates in the GA biosynthetic pathway revealed that the step from GA₁₉ to GA₂₀ was blocked in transgenic tobacco plants overexpressing NTH15 . To investigate the relationship between the expression of NTH15 and genes involved in GA biosynthesis, we isolated three cDNA clones from tobacco encoding two types of GA 20-oxidase and a 3β-hydroxylase. RNA gel blot analysis revealed that the expression of one gene ( Ntc12 , encoding GA 20-oxidase), which in wild-type tobacco plants was abundantly expressed in leaves, was strongly suppressed in the transformants. The expression level of Ntc12 decreased with increasing severity of phenotype of transgenic tobacco leaves. The abnormal leaf morphology was largely overcome by treatment with GA₂₀ or GA₁ but not by GA₁₉. These data strongly suggest that overexpression of NTH15 inhibits the expression of Ntc12 , resulting in reduced levels of active GA and abnormal leaf morphology in transgenic tobacco plants. In situ hybridization in wild-type tobacco revealed that expression of Ntc12 occurred mainly in the rib meristem, cells surrounding the procambium and in leaf primordia. Expression was not seen in the tunica, corpus and procambium, tissues in which NTH15 was predominantly expressed. The contrasting expression patterns of these genes may reflect their antagonistic functions in the formation of lateral organs from the shoot apical meristem.     

Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants

Degradation of active C(19)-gibberellins (GAs) by dioxygenases through 2beta-hydroxylation yields inactive GA products. We identified two genes in Arabidopsis (AtGA2ox7 and AtGA2ox8), using an activation-tagging mutant screen, that encode 2beta-hydroxylases. GA levels in both activation-tagged lines were reduced significantly, and the lines displayed dwarf phenotypes typical of mutants with a GA deficiency. Increased expression of either AtGA2ox7 or AtGA2ox8 also caused a dwarf phenotype in tobacco, indicating that the substrates for these enzymes are conserved. AtGA2ox7 and AtGA2ox8 are more similar to each other than to other proteins encoded in the Arabidopsis genome, indicating that they may constitute a separate class of GA-modifying enzymes. Indeed, enzymatic assays demonstrated that AtGA2ox7 and AtGA2ox8 both perform the same GA modification: 2beta-hydroxylation of C(20)-GAs but not of C(19)-GAs. Lines containing increased expression of AtGA2ox8 exhibited a GA dose-response curve for stem elongation similar to that of the biosynthetic mutant ga1-11. Double loss-of-function Atga2ox7 Atga2ox8 mutants had twofold to fourfold higher levels of active GAs and displayed phenotypes associated with excess GAs, such as early bolting in short days, resistance to the GA biosynthesis inhibitor ancymidol, and decreased mRNA levels of AtGA20ox1, a gene in the GA biosynthetic pathway.     

Overexpression of HBK3, a class I KNOX homeobox gene, improves the development of Norway spruce (Picea abies) somatic embryos

In order to investigate the effects of HBK3, a spruce gene member of the class I KNOX family, during somatic embryogenesis, sense (HBK3-S) and antisense (HBK3-A) Norway spruce (Picea abies) lines were generated. Somatic embryos produced from these lines were then analysed at morphological and structural levels. Compared with control, differentiation of immature somatic embryos from pro-embryogenic masses (PEMs) was accelerated in lines overexpressing HBK3 (HBK3-S). Such immature embryos showed enlarged embryogenic heads and were able to produce fully developed cotyledonary embryos at higher frequency. Furthermore, HBK3-S embryos had enlarged shoot apical meristems (SAMs) and enlarged expression pattern of PgAGO, a molecular marker gene specific to meristematic cells. Lines in which HBK3 (HBK3-A) was down-regulated had reduced ability to produce immature somatic embryos from PEMs and were not able to complete the maturation processes. To assess the function of HBK3 in comparison with that of angiosperm KNOX genes, this gene was ectopically expressed in Arabidopsis plants. As observed for spruce, Arabidopsis embryos overexpressing HBK3 had enlarged meristems and enlarged expression pattern of SHOOTMERISTEMLESS, a SAM molecular marker gene. In addition, transformed embryos were able to germinate at a higher rate and the resulting plants showed a variety of phenotypic aberrations, including abnormal leaves and reduced apical dominance. Overall, these data confirm the importance of KNOTTED genes during development and reveal the participation of HBK3 in conifer embryogeny. Furthermore, the results show redundant functions of this gene during embryonic growth of spruce and Arabidopsis, but not during post-embryonic growth.     

Overexpression and clinicopathological significance of homeobox gene Quox-1 in oral squamous cell carcinoma

The expression and clinicopathological significance of Quox-1 gene was studied in oral squamous cell carcinoma (OSCC). Immunocytochemistry and western blot analysis were used to examine the different expressions of Quox-1 protein in 114 OSCC specimens, 34 oral epithelial dysplasia specimens, and 16 normal oral mucosa specimens. RT-PCR and virtual Northern Blot were also used to examine the expression of Quox-1 mRNA. It was found that Quox-1 was not expressed in normal epithelium. However, as dysplastic lesions progressed Quox-1 expression increased (p < 0.01), and Quox-1 expression was not significantly different between severe dysplasia and highly differentiated OSCCs (p > 0.05). As the degree of differentiation decreased, Quox-1 positivity increased in OSCC (p < 0.01), and the rate of Quox-1 (81.58%) positivity in OSCC was higher than that in normal oral mucosa (p < 0.01). Our findings imply that the positive expression of Quox-1 is correlated with the histological classification of OSCCs. Thus, the expression of Quox-1 in OSCC may serve as a significant predicting factor of proliferative status and malignant degree, and it may also be a biological detection marker of oral mucosas initial cancer and of OSCC.     

Increased accumulation of anthocyanins in transgenic potato tubers by overexpressing the 3GT gene

In order to explore a biotechnological method for improving potato tuber color and creating plants with increased anthocyanin contents, a potato UDP-glucose: flavonoid-3-O-glucosyltransferase (3GT) gene was inserted behind the GBSSI promoter of pBin19, and this construct was introduced into Solanum tuberosum L. cultivar Désirée plants by Agrobacterium-mediated transformation. Six independent transgenic lines overexpressing the 3GT gene were identified by PCR and Southern blot analysis from 18 kanamycin-resistant plants. Due to the expression of 3GT gene, the tuber color and the anthocyanin content were enhanced noticeably in the transgenic plants compared to the wild-type control plants. This result suggests that the 3GT gene can potentially be used to improve potato color and enhance levels of antioxidants in the diet.     

Changes in jasmonate and gibberellin levels during development of potato plants (Solanum tuberosum)

Among the multiple environmental signals and hormonal factors regulatingpotato plant morphogenesis and controlling tuber induction, jasmonates (JAs)andgibberellins (GAs) are important components of the signalling pathways in theseprocesses. In the present study, with Solanum tuberosum L.cv. Spunta, we followed the endogenous changes of JAs and GAs during thedevelopmental stages of soil-grown potato plants. Foliage at initial growthshowed the highest jasmonic acid (JA) concentration, while in roots the highestcontent was observed in the stage of tuber set. In stolons at the developmentalstage of tuber set an important increase of JA was found; however, in tubersthere was no change in this compound during tuber set and subsequent growth.Methyl jasmonate (Me-JA) in foliage did not show the same pattern as JA; Me-JAdecreased during the developmental stages in which it was monitored, meanwhileJA increased during those stages. The highest total amount of JAs expressed asJA+Me-JA was found at tuber set. A very important peak ofJA in roots was coincident with that observed in stolons at tuber set. Also, aprogressive increase of this compound in roots was shown during the transitionof stolons to tubers. Of the two GAs monitored, gibberellic acid(GA₃) was the most abundant in all the organs. While GA₁and GA₃ were also found in stolons at the time of tuber set, noothermeasurements of GAs were obtained for stolons at previous stages of plantdevelopment. Our results indicate that high levels of JA and GAs are found indifferent tissues, especially during stolon growth and tuber set.     

Arx homeobox gene is essential for development of mouse olfactory system

The olfactory system provides an excellent model in which to study cell proliferation, migration, differentiation, axon guidance, dendritic morphogenesis, and synapse formation. We report here crucial roles of the Arx homeobox gene in the developing olfactory system by analyzing its mutant phenotypes. Arx protein was expressed strongly in the interneurons and weakly in the radial glia of the olfactory bulb, but in neither the olfactory sensory neurons nor bulbar projection neurons. Arx-deficient mice showed severe anatomical abnormalities in the developing olfactory system: (1) size reduction of the olfactory bulb, (2) reduced proliferation and impaired entry into the olfactory bulb of interneuron progenitors, (3) loss of tyrosine hydroxylase-positive periglomerular cells, (4) disorganization of the layer structure of the olfactory bulb, and (5) abnormal axonal termination of olfactory sensory neurons in an unusual axon-tangled structure, the fibrocellular mass. Thus, Arx is required for not only the proper developmental processes of Arx-expressing interneurons, but also the establishment of functional olfactory neural circuitry by affecting Arx-non-expressing sensory neurons and projection neurons. These findings suggest a likely role of Arx in regulating the expression of putative instructive signals produced in the olfactory bulb for the proper innervation of olfactory sensory axons.