A maize vacuolar invertase, IVR2, is induced by water stress. Organ/tissue specificity and diurnal modulation of expression.

The expression of invertases was analyzed in vegetative organs of well-watered and water-stressed maize (Zea mays) plants. Early changes in sucrose metabolism and in acid soluble invertase expression were observed in vegetative sink and source organs under mild water stress. The organ-specific induction of acid invertase activity was correlated with an increase in the Ivr2 gene transcripts and in the vacuolar invertase proteins. In addition diurnal changes in activity and Ivr2 transcripts for vacuolar invertase were noted in shoots. Hexoses (glucose and fructose) accumulated in all organs examined from water-stressed plants. In situ localization studies showed that glucose accumulation, vacuolar invertase activity, invertase protein, and the Ivr2 transcripts colocalized specifically in bundle sheath and vascular tissue cells of mature stressed leaf; in primary roots the stress-induced increase of Ivr2 transcripts was detected only in root tips. Based on these results different regulatory roles are proposed in sink and source organs for the stress induced Ivr2 vacuolar invertase.

Defective splicing of the first nad4 intron is associated with lack of several complex I subunits in the Nicotiana sylvestris NMS1 nuclear mutant.

In this work, we provide evidence for the existence of a nuclear factor involved in the splicing of a specific mitochondrial intron in higher plants. In the Nicotiana sylvestris nuclear NMS1 mutant, defective in both vegetative and reproductive development, the first intron of the nad4 transcript encoding the complex I NAD4 subunit is not removed, whatever the tissue analysed. Transcript patterns of other standard mitochondrial genes are not affected in NMS1. However, numerous polypeptides are missing in two-dimensional in organelle mitochondrial protein synthesis patterns and several nuclear and mitochondrial complex I subunits are present in trace amounts. This indicates that translational or post-translational steps in the synthesis of other mitochondrial proteins are affected. All of these defects co-segregated with the abnormal phenotype in the offspring of a NMS1 x wild-type cross, showing that they are controlled by the same nuclear gene (MS1) or tightly linked loci. Such a complex situation has been described in chloroplasts and mitochondria of fungi, but never in higher plant mitochondria.

Characterization of two members of the maize gene family, Incw3 and Incw4, encoding cell-wall invertases.

Two maize putative cell-wall invertase genes (Incw3 and Incw4) have been isolated by screening a genomic DNA library (Zea mays L. W22) using the cDNA probes encoding the two maize cell-wall invertases Incw1 and Incw2. The Incw3 and Incw4 genes contain six exons/five introns and five exons/four introns, respectively. The protein sequences deduced from both genes revealed a beta-fructosidase motif and a cysteine catalytic site known to be conserved in invertase genes. A detailed analysis of the protein and nucleotide sequences provides evidence that the Incw3 and the Incw4 genes encode putative cell-wall invertases. Furthermore, the isoelectric point deduced from the INCW4 protein sequence suggested that the Incw4 gene may encode a unique type of cell-wall invertase unbound in the apoplast. Gene expression studies using RT-PCR and in-situ RT-PCR hybridization showed that the Incw3 expression is organ/tissue-specific and developmentally regulated. In contrast, the Incw4 gene is constitutively expressed in all vegetative and reproductive tissues tested.

Forcing expression of a soybean root glutamine synthetase gene in tobacco leaves induces a native gene encoding cytosolic enzyme.

Glutamine synthetase (GS; EC 6.3.1.2) is present in different subcellular compartments in plants. It is located in the cytoplasm in root and root nodules while generally present in the chloroplasts in leaves. The expression of GS gene(s) is enhanced in root nodules and in soybean roots treated with ammonia. We have isolated four genes encoding subunits of cytosolic GS from soybean (Glycine max L. cv. Prize). Promoter analysis of one of these genes (GS15) showed that it is expressed in a root-specific manner in transgenic tobacco and Lotus corniculatus, but is induced by ammonia only in the legume background. Making the GS15 gene expression constitutive by fusion with the CaMV-35S promoter led to the expression of GS in the leaves of transgenic tobacco plants. The soybean GS was functional and was located in the cytoplasm in tobacco leaves where this enzyme is not normally present. Forcing this change in the location of GS caused concomitant induction of the mRNA for a native cytosolic GS in the leaves of transgenic tobacco. Shifting the subcellular location of GS in transgenic plants apparently altered the nitrogen metabolism and forced the induction in leaves of a native GS gene encoding a cytosolic enzyme. The latter is normally expressed only in the root tissue of tobacco. This phenomenon may suggest a hitherto uncharacterized metabolic control on the expression of certain genes in plants.

Ultrastructural detection of ribulose-1,5-bisphosphate carboxylase protein and its subunit mRNAs in wild-type and holoenzyme-deficient Nicotiana using immuno-gold and in-situ-hybridization techniques.

In-situ-localization techniques have been adapted to the ultrastructural detection of the holoenzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) and its composite large- and smallsubunit mRNAs in wild-type and mutant RuBPCase deficient plantlets of Nicotiana tabacum L. Immuno-gold techniques which show the distribution of target proteins have confirmed visually the presence of the holoenzyme in the wild-type plastids and its total absence in the enzyme-less mutant. Using in-situ hybridization coupled with electron microscopy and biotinylated probes for the two subunits, we have directly visualized specific small-subunit mRNAs located in the cytoplasm and large-subunit mRNAs confined to plastids in the enzyme-deficient mutant, and with apparent distributions comparable to those visualized in the wild-type counterpart. These results show that (i) gene products can be visualized in situ by electronmicroscopy techniques under conditions where the respective cellular compartments are readily recognizable and (ii) that an accumulation of mRNAs corresponding to the composite subunits can occur without translation and-or assembly of the protein.

Localization of mRNAs for the Small and Large Subunits of Rubisco Using Electron Microscopic in Situ Hybridization.

In situ hybridization coupled with electron microscopy has been used to locate mRNAs for the small and large subunits of ribulose 1,5-bisphosphate carboxlase in young leaf tissue of tobacco (Nicotiana tabacum L.) plants. The endogeneous mRNAs were hybridized with either a biotinylated DNA probe for the small subunit or large subunit and subsequently visualized using avidin-ferritin conjugates at the electron microscope level. In the tissue incubated with the small subunit cDNA probe, the cytoplasm was uniformly labeled with ferritin indicating the presence of the target mRNA; this was particularly visible in cells which had under-gone some structural damage. In the case of the LSU probe, the ferritin marker was shown to be exclusively associated with the plastid stroma in intact leaf cells. The compartmentation of cytoplasmic small subunit mRNA versus plastid large subunit mRNA has been confirmed by direct visualization of in situ hybridization.

Terpenoid Metabolism in Plastids : Isolation and Biochemical Characteristics of Capsicum annuum Chromoplasts.

A technique for the isolation and the purification of Capsicum annum L. var. Yolo Wonder chromoplasts is described. The degree of purity of the isolated chromoplasts is greatly improved by the absence of MgCl(2) in the extraction medium and in the gradient purification system, as shown by electron micrographs and the near absence of antimycin-insensitive NADH:cytochrome c reductase activity and succinate:cytochrome c reductase activity. Furthermore, phosphatidylserine was absent and the phosphatidylethanolamine content was reduced by a factor of 5 in these preparations, which were active in the synthesis of galactolipids, prenylquinones, and carotenoids.

Carotenoid metabolism during chloroplast to chromoplast transformation in Capsicum annuum fruit.

Lipid and carotenoid metabolism associated with the structural transformations of the plastids during the ripening of Capsicum annuum fruits were studied. The early ripening stage is characterized by chloroplasts with a typical grana-intergranal structure and a highly developed peripheral reticulum. At a later stage the thylakoid system is disintegrated and replaced by non-chlorophyllous single thylakoids, derived in part from the inner envelope membrane. First, these changes coincide with a loss of galactolipids, a slow increase in phospholipid content, a decrease in the ratio galactolipids/phospholipids and in the galactosyl transferase activities on a protein basis. Second, these changes correlate with an enhanced accumulation of keto-carotenoids. When the phospholipid environment is disturbed by difluorodinitrobenzene or phospholipases, the biosynthesis of phytoene, ß-carotene, and capsanthin is inhibited; phospholipase D has a greater effect. The role of a phospholipid environment in carotenoid biosynthesis is discussed.

Interaction between External and Internal Conditions in the Development of Photosynthetic Features in a Grass Leaf: I. REGIONAL RESPONSES ALONG A LEAF DURING AND AFTER LOW-LIGHT OR HIGH-LIGHT ACCLIMATION.

Morphological and functional features were compared along a developing third leaf and fully expanded leaf from high-light- and low-light-acclimated seedlings of Lolium multiflorum.The young leaf contains a gradient of differentiating tissue, ranging from meristematic cells at the leaf base to mature tissue at the tip; this gradient can be related to the maturation of a functional photosynthetic apparatus. Along the fully expanded leaf, a decreasing gradient from tip to base is maintained for functional characteristics (net maximum photosynthesis, chlorophyll content, and ribulose bisphosphate carboxylase activity) and for a number of structural parameters (number of mesophyll cells and their external surface area, number of chloroplasts and their envelope area), irrespective of the light regime. In contrast, a constancy in the absolute intrachloroplastic lamellar content per plastid was revealed whatever the position in the leaf or irradiance received. However, the relative membrane content was lower in high-light chloroplasts due to their larger volume compared to low-light plastids (dilution effect).The longitudinal differences in functional and morphological characteristics are interpreted as the result of interaction between the internal gradient of differentiating tissue along a developing young leaf and the external light conditions during development.

Interaction between External and Internal Conditions in the Development of Photosynthetic Features in a Grass Leaf: II. REVERSIBILITY OF LIGHT-INDUCED RESPONSES AS A FUNCTION OF DEVELOPMENTAL STAGES.

Lolium multiflorum plants were grown under a low- or high-light regime until third leaves had emerged to one-third their final length and then were transferred to a contrasting light regime. At this stage, the leaf possesses a tissue-age gradient from tip to base; thus, the reversibility of light-acclimated responses as a function of the degree of differentiation was analyzed in individual leaves.Regional responses in the apical, medial, and basal zones of leaves of transferred plants were analyzed before and after light transfer and compared to leaves from plants kept in constant light. Leaves transferred from low to high light showed rapid recovery and attained light-saturated rates and ribulose bisphosphate carboxylase activities equivalent to those of high-light controls. However, fresh or dry weight and chlorophyll content were intermediate between those for the two irradiances. In the reciprocal experiment, the apical leaf zone retained high-light characteristics for maximum photosynthesis, whereas all the other functional parameters adapted rapidly to values characteristic of their low-light counterparts (equivalent foliar zones).On the ultrastructural level, chloroplasts in the apical zone of transferred leaves surpassed their respective constant light controls in absolute membrane content and size. However, a shift to high light induced an increase in plastid volume and, in relative terms, the plastid membrane content was diluted. A shift to low-light treatment led to smaller membrane-dense plastids. The ultrastructural readaptation is realized through differential rates of increase in plastid volume and lamellar content. Proplastids (leaf base), or juvenile plastids having reached an intermediate developmental stage (leaf middle zone) at the time of transfer, took on characteristics of the latter light regime (equivalent to controls).These results provide evidence for rapid reacclimation processes under changing light regimes and suggest a capacity for regional light responses along the leaf.