Structure and immunocytochemical localization of photosynthetic enzymes in the lamina joint and sheath pulvinus of the C4 grass Arundinella hirta.

The C(4) grass Arundinella hirta exhibits a unique C(4) anatomy, with isolated Kranz cells (distinctive cells) and C(4)-type expression of photosynthetic enzymes in the leaf sheath and stem as well as in the leaf blade. The border zones between these organs are pale green. Those between the leaf blade and sheath and between the sheath and stem are called the lamina joint and sheath pulvinus, respectively, and are involved in gravity sensing. We investigated the structure and localization of C(3) and C(4) photosynthetic enzymes in these tissues. In both zones the epidermis lacked stomata. The inner tissue was composed of parenchyma cells and vascular bundles. The parenchyma cells were densely packed with small intercellular spaces and contained granal chloroplasts with large starch grains. No C(4)-type cellular differentiation was recognized. Western blot analysis showed that the lamina joint and pulvinus accumulated substantial amounts of phosphoenolpyruvate carboxylase (PEPC), pyruvate,Pi dikinase (PPDK), and ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco). Immunogold electron microscopy revealed PEPC in the cytosol and both PPDK and rubisco in the chloroplasts of parenchyma cells, suggesting the occurrence of C(3) and C(4) enzymes within a single type of chlorenchyma cell. These data indicate that the lamina joint and pulvinus have unique expression patterns of C(3) and C(4) enzymes, unlike those in C(4)-type anatomy.

Further examination of abscission zone cells as ethylene target cells in higher plants.

BACKGROUND AND AIMS: Two aspects of the competence of abscission zone cells as a specific class of hormone target cell are examined. The first is the competence of these target cells to respond to a remote stele-generated signal, and whether ethylene acts in concert with this signal to initiate abscission of the primary leaf in Phaseolus vulgaris. The second is to extend the concept of dual control of abscission cell competence. Can the concept of developmental memory that is retained by abscission cell of Phaseolus vulgaris post-separation in terms of the inductive/repressive control of beta-1,4-glucan endohydrolase (cellulase) activity exerted by ethylene/auxin be extended to the rachis abscission zone cells of Sambucus nigra? METHODS: Abscission assays were performed using the leaf petiole-pulvinus explants of P. vulgaris with the distal pulvinus stele removed. These (-stele) explants do not separate when treated with ethylene and require a stele-generated signal from the distal pulvinus for separation at the leaf petiole-pulvinis abscission zone. Using these explants, the role of ethylene was examined, using the ethylene action blocker, 1-methyl cyclopropene, as well as the significance of the tissue from which the stele signal originates. Further, leaf rachis abscission explants were excised from the compound leaves of S. nigra, and changes in the activity of cellulase in response to added ethylene and auxin post-separation was examined. KEY RESULTS: The use of (-stele) explants has confirmed that ethylene, with the stele-generated signal, is essential for abscission. Neither ethylene alone nor the stelar signal alone is sufficient. Further, in addition to the leaf pulvinus distal to the abscission zone, mid-rib tissue that is excised from senescent or green mid-rib tissue can also generate a competent stelar signal. Experiments with rachis abscission explants of S. nigra have shown that auxin, when added to cells post-separation can retard cellulase activity, with activity re-established with subsequent ethylene treatment. CONCLUSIONS: The triggers that initiate and regulate the separation process are complex with, in bean leaves at least, the generation of a signal (or signals) from remote tissues, in concert with ethylene, a requisite part of the process. Once evoked, abscission cells maintain a developmental memory such that the induction/repression mediated by ethylene/auxin that is observed prior to separation is also retained by the cells post-separation.

Bioorganic studies on plant movement, from natural products to its receptor.

The chemical aspects of the circadian leaf movement known as "nyctinasty" are discussed in this paper. Each of the nyctinastic plants of five different genera so far examined contained a pair of factors, one of which induced leaf closure and another induced leaf opening. The relative contents of the closing and opening factors changed correlating with the nyctinastic leaf movement. The use of fluorescence-labeled and photoaffinity-labeled factors revealed that the factors bind to specific cells, the motor cells, present in the pulvini, and that the membrane fraction of the motor cells contained two proteins of 210 and 180 kDa, which can bind to the factors.

The enzyme involved in sulfation of the turgorin, gallic acid 4-O-(beta-D-glucopyranosyl-6'-sulfate) is pulvini-localized in Mimosa pudica.

A sulfotransferase (ST) which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gallic acid glucoside was characterized from microsomal preparations of Mimosa pudica. The product of the reaction was found to co-elute on HPLC with the periodic leaf movement factor 1 (PLMF-1)(gallic acid beta-D-gluco-pyranosyl-6'-sulfate). The distribution of the enzyme activity was restricted to plasma membrane preparations from primary, secondary and tertiary pulvini. The M. pudica ST activity was inhibited in a dose-dependent manner in the presence of an antibody raised against the flavonol 3-sulfotransferase of Flaveria chloraefolia, suggesting structural similarities between the two proteins. Western blot analysis of M. pudica protein extracts using these antibodies indicated the presence of a cross-reactive polypeptide with an apparent molecular mass of 42,000 Da whose distribution correlates with the presence of the gallic acid glucoside ST activity. Indirect immunogold labeling of resin-embedded sections from tertiary pulvini showed a specific localization of gold particles on the sieve-tube plasma membranes. The label distribution was uniform and other cellular organelles and membrane systems displayed little or no labeling. The results of the Western blot and immunocytochemical studies are consistent with the detection of the gallic acid glucoside ST activity in plasma membrane preparations of M. pudica pulvini cells. The specific tissue distribution of the ST in motor organ phloem cells suggests that this is the site of synthesis and/or accumulation of PLMF-1 and supports the proposed hypothesis that PLMF-1 may be acting as a chemical signal during the seismonastic response of M. pudica.

Molecular basis of the increase in invertase activity elicited by gravistimulation of oat-shoot pulvini.

An asymmetric (top vs. bottom) increase in invertase activity is elicited by gravistimulation in oat-shoot pulvini starting within 3 h after treatment. In order to analyze the regulation of invertase gene expression in this system, we examined the effect of gravistimulation on invertase mRNA induction. Total RNA and poly (A)+RNA, isolated from oat pulvini, and two oligonucleotide primers, corresponding to two conserved amino-acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial-length cDNA (550 base pairs) was obtained and characterized. There was a 52% deduced amino-acid sequence homology to that of carrot beta-fructosidase and a 48% homology to that of tomato invertase. Northern blot analysis showed that there was an obvious transient accumulation of invertase mRNA elicited by gravistimulation of oat pulvini. The mRNA was rapidly induced to a maximum level at 1 h following gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher (five-fold) than that in the top half of the pulvinus tissue. The induction of invertase mRNA was consistent with the transient enhancement of invertase activity during the graviresponse of the pulvinus. These data indicate that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional and/or translational levels. Southern blot analysis showed that there were four genomic DNA fragments hybridized to the invertase cDNA. This suggests that an invertase gene family may exist in oat plants.

Cell wall and enzyme changes during the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa).

The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response accompanied by several asymmetric processes, including degradation of starch and cell wall synthesis. To understand further the cellular and biochemical events associated with the graviresponse, changes in cell walls and their constituents and the activities of related enzymes were investigated in excised pulvini. Asymmetric increases in dry weight with relatively symmetric increases in wall weight accompanied the graviresponse. Starch degradation could not account for increases in wall weight. However, a strong asymmetry in invertase activity indicated that hydrolysis of exogenous sucrose could contribute significantly to the increases in wall and dry weights. Most cell wall components increased proportionately during the graviresponse. However, beta-D-glucan did not increase symmetrically, but rather increased in proportion in lower halves of gravistimulated pulvini. This change resulted from an increase in glucan synthase activity in lower halves. The asymmetry of beta-D-glucan content arose too slowly to account for initiation of the graviresponse. A similar pattern in change in wall extensibility was also observed. Since beta-D-glucan was the only wall component to change, it is hypothesized that this change is the basis for the change in wall extensibility. Since wall extensibility changed too slowly to account for growth initiation, it is postulated that asymmetric changes in osmotic solutes act as the driving factor for growth promotion in the graviresponse, while wall extensibility acts as a limiting factor during growth.