Ultrastructural detection of sucrose synthase distribution in developing maize leaves

Summary A developing maize leaf grows by the activity of a basal meristematic region and an adjacent elongating zone, resulting in a morphological and functional gradient along the leaf. We have used this system to detect the spatial and temporal expression of an enzyme, sucrose synthase, which plays a pivotal role in the sucrose import-export transition which occurs along a monocotyledon leaf. Immunogold labeling was used to detect the cellular and sub-cellular distribution of sucrose synthase (SS) at the electron microscopical level; the protein was visualized using a polyclonal antiserum on embedded tissue sections. Immunolabel was observed in the cytosol of dividing meristematic cells, expanding cells of the elongation zone, and in differentiating cells of young photosynthetic tissue. In fully differentiated leaf tissue, however, the protein was no longer immuno-detectable in photosynthetic cells, but was present in the guard and subsidiary cells of stomata and in companion cells within the phloem tissue of vascular bundles. The tissue- and cell-specific localization of sucrose synthase changes along the growing leaf as a function of the developmental state and the associated need for sucrose import or export.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

Upstream regulatory regions from the maize Sh1 promoter confer tissue-specific expression of the ,-glucuronidase gene in tomato

A promoter fusion (Sh35) combining upstream regulatory regions from the maize Sh1 promoter with a truncated 35S promoter, Δ9035 (–90 to +8) has been compared with the original Sh1 promoter for its capacity to promote expression of the β-glucuronidase (GUS) gene in stably transformed tomato plants. For both promoters, very faint GUS expression was detected in the vegetative tissues, and no expression was detected in the fruit pericarp tissues. However, in the seed, Sh1 promoted low GUS expression but Sh35 directed 25-fold higher GUS expression. For both constructs, the profile of GUS expression was similar to that of endogenous sucrose synthase activity, but maximal GUS activity was reached 15 days after the peak of sucrose synthase activity. Received: 20 October 1998 / Revision received: 1 December 1998 / Accepted: 14 December 1998     

Co-suppression in transgenic Petunia hybrida expressing chalcone synthase A ( chsA )

Chalcone synthase A is a key enzyme in the anthocyanin biosynthesis pathway. Expression ofchsA gene in transgenicPetunia hybrida resulted in flower color alterations and co-suppression of transgenes and endogenous genes. We fused the β-glucuronidase (uidA) gene to the C-terminal ofchsA gene, and transferred the fusion gene intoPetunia hybrida viaAgrobacterium tumefaciens. GUS histochemical staining analysis showed that co-suppression occurred specifically during the development of flowers and co-suppression required the mutual interaction of endogenous genes and transgenes. RNAin situ hybridization analysis suggested that co-suppression occurred in the entire plant, and RNA degradation occurred in the cytoplasm.     

Differential expression profiles of growth-related genes in the elongation zone of maize primary roots

Growth in the apical elongation zone of plant roots is central to the development of functional root systems. Rates of root segmental elongation change from accelerating to decelerating as cell development proceeds from newly formed to fully elongated status. One of the primary variables regulating these changes in elongation rates is the extensibility of the elongating cell walls. To help decipher the complex molecular mechanisms involved in spatially variable root growth, we performed a gene identification study along primary root tips of maize (Zea mays) seedlings using suppression subtractive hybridization (SSH) and candidate gene approaches. Using SSH we isolated 150 non-redundant cDNA clones representing root growth-related genes (RGGs) that were preferentially expressed in the elongation zone. Differential expression patterns were revealed by Northern blot analysis for 41 of the identified genes and several candidate genes. Many of the genes have not been previously reported to be involved in root growth processes in maize. Genes were classified into groups based on the predicted function of the encoded proteins: cell wall metabolism, cytoskeleton, general metabolism, signaling and unknown. In-situ hybridization performed for two selected genes, confirmed the spatial distribution of expression shown by Northern blots and revealed subtle differences in tissue localization. Interestingly, spatial profiles of expression for some cell wall related genes appeared to correlate with the profile of accelerating root elongation and changed appropriately under growth-inhibitory water deficit.     

Sequence and expression analysis of a Xenopus laevis cDNA which encodes a homologue of mammalian 14-3-3 zeta protein

We report the cloning and characterisation of a cDNA that encodes a novel member of the Xenopus laevis 14-3-3 protein family. Sequence analysis reveals that the cDNA-encoded protein shares 84% identity with the rat, human or sheep 14-3-3ζ isoform, and between 66% and 77% identity with bovine, human or rat β, bovine γ, human τ, Drosophila 14-3-3 and a previously isolated Xenopus member. The corresponding mRNA is present in all adult tissues examined with the highest levels in the brain. Although the gene is expressed throughout embryogenesis, higher levels of mRNA accumulate after gastrulation. Whole-mount in situ hybridisation on tailbud stage embryo reveals strong expression of the gene in the head, optic vesicles, spinal cord and branchial arches with weaker expression in the somites. In addition, expression along the notochord is observed at stage 45 (tadpole). This spatial and temporal expression profile along with recent studies implicating the importance of 14-3-3 proteins in the regulation of signal transduction pathways argues for a key role of this isoform in embryonic development.     

Influenza-B-virus-induced eye and brain malformations during early chick embryogenesis and localization of the viral RNA in specific areas

Influenza is prevalent worldwide, and the teratogenic effects of influenza infection have been suspected to occur within the developing central nervous system. We herein report the sequelae of influenza B viral infection during early chick embryogenesis. Chick embryos at Hamburger-Hamilton stage 9 were infected by an in ovo injection under the blastoderm of influenza B virus (B/Taiwan/25/99). At 48 h after infection, gross malformations of the eye and brain, ranging from 25 to 58% of 168 infected embryos, were observed, in contrast to 3–6% among 71 mock-infected controls (p < 0.0001 for both eye and brain malformations). Histological analyses showed extensive tissue degeneration and aggregates of cells in the head mesenchyme, suggesting cell death and heterotopia. Influenza B viral RNA was directly localized by in situ hybridization with probes specific for the HA segment. Viral RNA was extensively detected in the head surface ectoderm and in the lung bud. In the developing brain, viral RNA was specifically located in the anterior neural retina, habenular area, mid-thalamus, and rhombencephalon. Our data show that influenza B virus can be a teratogenic agent in neural and nonneural embryonic tissues, raising concern for transplacental infection during early pregnancy.     

In situ localization of male germ cell-associated kinase (mak) mRNA in adult mouse testis: specific expression in germ cells at stages around meiotic cell division.

Biochemical analysis of the male germ cell-associated kinase (mak) gene, which was isolated recently by using weak cross-hybridization with the v-ros tyrosine kinase gene, revealed that the gene was highly expressed in mammalian testicular germ cells, but not in ovarian cells. In order to identify the cells which express the mak gene in more detail, we localized mak mRNA in frozen sections of mouse testis by non-radioactive in situ hybridization. In this study, we utilized thymine-thymine (T-T) dimerized mak cDNA as a haptenic, non-radioactive probe, and the signal was detected enzyme-immunohistochemically by using an anti-T-T antibody. As a result, mak mRNA was localized intensely in late pachytene (stage X) and diplotene (stage XI) spermatocytes, and faintly in dividing spermatocytes (stage XII) and early round spermatids (stage I-II), suggesting that the gene may play an important role in the phase around meiotic cell division, but not throughout the entire meiosis.     

Cellular expression of murine Ym1 and Ym2, chitinase family proteins,as revealed by in situ hybridization and immunohistochemistry

Ym is one of the chitinase family proteins, which are widely distributed in mammalian bodies and can bind glycosaminoglycans such as heparin/heparan sulfate. Ym1 is a macrophage protein produced in parasitic infections, while its isoform, Ym2, is upregulated in lung under allergic conditions. In the present study, we revealed the distinct cellular expression of Ym1 and Ym2 in normal mice by in situ hybridization and immunohistochemistry. Ym1 was principally expressed in the lung, spleen, and bone marrow, while Ym2 was found in the stomach. Ym1-expressing cells in the lung were alveolar macrophages, and the immunoreactivity for Ym1 was localized in rough endoplasmic reticulum. In the spleen, Ym1-expressing cells gathered in the red pulp and were electron microscopically identified as immature neutrophils. In the bone marrow, immature neutrophils were intensely immunoreactive, but lost this immunoreactivity with maturation. Moreover, needle-shaped crystals in the cytoplasm of macrophages, which formed erythroblastic islands, also showed intense Ym1 immunoreactivity. Ym2 expression was restricted to the stratified squamous epithelium in the junctional region between forestomach and glandular stomach. The function of Ym1 and Ym2 is still unclear; however, the distinct cellular localization under normal conditions suggests their important roles in hematopoiesis, tissue remodeling, or immune responses as an endogenous lectin.     

Cloning, expression and processing of the CP2 neuropeptide precursor of Aplysia

The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.