Plastid transcription activity and DNA copy number were quantified during chloroplast development in the first foliage leaf in dark-grown and illuminated barley (Hordeum vulgare L.) seedlings. Primary foliage leaves of seedlings given continuous illumination from 2 days post-imbibition reached a final mean length of 15 centimeters at 6.5 days, whereas primary leaves of darkgrown seedlings required 7 days to reach a similar length. Dividing cells were observed in the basal 0.5 to 1 centimeter of primary leaves until 5.5 days post-imbibition. Plastids isolated from cells located in the basal meristem of 4-day-old seedlings were small (-2 micrometers in diameter), exhibited low transcription activity and contained approximately 130 copies of plastid DNA per organelle. Cell size increased from 18 to 60 micrometers in a 1 to 3 centimeter region located adjacent to the leaf basal meristem. In this region, transcriptional activity per plastid increased 10-fold and DNA copy number increased from 130 to 210. Plastid transcriptional activity declined rapidly in illuminated plants with increasing leaf cell age and plastid DNA copy number also declined but with a slower time course. In dark-grown seedlings, plastid transcriptional activity declined more slowly than in illuminated plants while DNA copy number remained constant with increasing cell age. These data show that plastid transcriptional activity and DNA copy number increase early in chloroplast development and that transcriptional activity per DNA template varies up to 5-fold during barley leaf biogenesis.In monocots such as barley or wheat, leaf cells are produced primarily by a meristem located in the leaf base (5). The meristematic cells of the leaf base contain small (1-2 ,um diameter) undifferentiated prochloroplasts (26). In contrast, mesophyll cells located in mature portions of barley leaves contain up to 60 chloroplasts which are 6 to 8 Am in diameter (12,26

Chloroplast genomes encode rRNAs, tRNAs, and proteins involved in transcription, translation, and photosynthesis. l h e expression of 15 plastid genes representing each of these functions was quantitated during chloroplast development in barley (Hordeum vulgare). l h e transcription of all plastid genes increased during the initial phase of chloroplast development and then declined during chloroplast maturation. RNAs corresponding to rpoBrpoC1-rpoC2, which encode subunits of a plastid RNA polymerase, and rpsl6, which encodes a ribosomal protein, reached maximal abundance early in chloroplast development prior to genes encoding subunits of the photosynthetic apparatus (rbcl, afp6, psaA, petB). lranscription of rpoB as well as 16s rRNA, trnfM-tmC, and trnK was high early in chloroplast development and declined 10-fold relative to r b c l transcription during chloroplast maturation. RNA hybridizing to psbA and psbD, genes encoding reaction center proteins of photosystem II, was differentially maintained in mature chloroplasts of illuminated barley. Differential accumulation of psbD mRNA relative to r b c l mRNA was due to light-stimulated transcription of psbD. In contrast, enhanced levels of psbA mRNA in mature chloroplasts were due primarily to selective stabilization of the psbA mRNA. These data document dynamic modulation of plastid gene transcription and mRNA stability during barley chloroplast development.

The mammalian CNS lacks the ability to effectively compensate for injury by the regeneration of damaged axons or axonal plasticity of intact axons. However, reports suggest that molecular or cellular manipulations can induce compensatory processes that could support regeneration or plasticity after trauma. We tested whether local, sustained release of the neurotrophic factor neurotrophin-3 (NT-3) would support axonal plasticity in the spinal cord distal to the site of injury in rats. The corticospinal tract (CST) was cut unilaterally at the level of the medulla. This avoided excessive inflammation, secondary cell death, vascular disruption, and the release of inhibitory molecules in the lumbar spinal cord. A replication-defective adenoviral vector (Adv) carrying the NT-3 gene (Adv.EFalpha-NT3) was delivered to the spinal motoneurons by retrograde transport through the sciatic nerve. Retrograde transport of the adenoviral vectors avoided the inflammatory response that would be associated with direct injection into the spinal cord. Transduction of spinal motoneurons with Adv.EFalpha-NT3 resulted in a significant increase in the concentration of NT-3 in the L3-L6 region of the spinal cord for up to 3 weeks. In animals with a CST lesion, this local expression of NT-3 induced growth of axons from the intact CST across the midline to the denervated side. If the CST remained intact, overexpression of NT-3 did not lead to an increase in the number of axons crossing the midline. These data demonstrate that local, sustained expression of NT-3 will support axonal plasticity of intact CST axons after trauma-induced denervation.

Application of neurotrophic factors (NFs) to the cut stump of motor nerves of neonatal rats confers neuroprotection from trauma-induced neuronal death. To test whether motoneurons are capable of responding to endogenously produced NFs, facial motoneurons were genetically modified in vivo to express several NFs and then tested for their response to peripheral nerve damage. Replication-defective adenoviral vectors [Adv.Rous sarcoma virus (RSV)-nf] representing three families of NFs were constructed that carried genes for brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor. Media from cultured cells transduced with Adv.RSV-nf contained NFs that supported the survival of cultured chick sensory neurons in the same manner as recombinant NF standards. When Adv.RSV-nf or an adenoviral vector containing the ␤-galactosidase gene (Adv.RSV-␤-gal) were injected into the facial muscles of neonatal rats the vectors were retrogradely transported to the facial nucleus where the NFs or ␤-gal were expressed. A fraction (ϳ10%) of the neurons were transduced as demonstrated by reverse transcriptase-PCR, histochemistry, and immunocytochemistry. In the case of Adv.RSV-BDNF, Adv.RSV-CNTF, and Adv.RSV-GDNF, a significant portion of the facial nucleus neurons was protected, 16.5, 18.2, and 53.3%, respectively, from death after axotomy, showing that neurons are capable of transporting the Adv.RSV-nf, expressing the recombinant NF genes, and responding to the NFs. In the case of Adv.RSV-GDNF, a greater number of facial nucleus motoneurons survived than were transduced, indicating that neighboring untransduced neurons were protected by the GDNF expressed by the transduced neurons by a paracrine mechanism.

No abstract