Andrzej K. Kononowicz

Regulation of the Osmotin Gene Promoter.

By introducing a chimeric gene fusion of the osmotin promoter and [beta]-glucuronidase into tobacco by Agrobacterium-mediated transformation, we have demonstrated a very specific pattern of temporal and spatial regulation of the osmotin promoter during normal plant development and after adaptation to NaCl. We have found that the osmotin promoter has a very high natural level of activity in mature pollen grains during anther dehiscence and in pericarp tissue at the final, desiccating stages of fruit development. GUS activity was rapidly lost after pollen germination. The osmotin promoter thus appears to be unique among active pollen promoters described to date in that it is active only in dehydrated pollen. The osmotin promoter was also active in corolla tissue at the onset of senescence. Adaptation of plants to NaCl highly stimulated osmotin promoter activity in epidermal and cortex parenchyma cells in the root elongation zone; in epidermis and xylem parenchyma cells in stem internodes; and in epidermis, mesophyll, and xylem parenchyma cells in developed leaves. The spatial and temporal expression pattern of the osmotin gene appears consistent with both osmotic and pathogen defense functions of the gene.

A higher plant extracellular vitronectin-like adhesion protein is related to the translational elongation factor-1 alpha.

Higher plant proteins immunologically related to the animal substrate adhesion molecule vitronectin have recently been observed and implicated in a variety of biological processes, such as plasma membrane-cell wall adhesion, pollen tube extension, and bacterium-plant interaction. We provide evidence that, similar to vitronectin, one of these proteins, PVN1 (plant vitronectin-like 1), isolated from 428 mM NaCl-adapted tobacco cells binds to glass surfaces an heparin. PVN1 was isolated by glass bead affinity chromatography. Isolated PVN1 has adhesive activity based on results from a baby hamster kidney cell-spreading assay. This plant adhesion protein was detected in all tissues examined but was most abundant in roots and salt-adapted cultured cells. Immunogold labeling indicated that PVN1 is localized in the cell wall of cortical and transmitting tissue cells of pollinated mature styles. A partial amino acid sequence of PVN1 revealed no similarity with vitronectin but, instead, was nearly identical to the translational elongation factor-1 alpha (EF-1 alpha). A clone isolated by screening a tobacco cDNA expression library with anti-PVN1 encoded a protein with greater than 93% identity to sequences of EF-1 alpha from plants of numerous species. Immunological cross-reactivity between tobacco PVN1 and EF-1 alpha as well as the reaction between the EF-1 alpha antibody and the 65- and 75-kD vitronectin-like proteins of a fucoidal alga supported the conclusion that the plant extracellular adhesion protein PVN1 is related to EF-1 alpha.

Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences

SummaryTransgenic sorghum plants (Sorghum bicolor L. Moench, cv. SRN39) were obtained by microprojectile-mediated DNA delivery (Bio-Rad PDS 1000/He Biolistic Delivery System) to explants derived from immature inflorescences. Explants were precultured on medium supplemented with 2.5 mg/l (11.31 µM) 2,4-D, 0.5 mg/l (2.32 µM) kinetin, and 60 g/l sucrose for 1 to 2 wk prior to bombardment. Bialaphos selectron pressure was imposed 2 wk after bombardment and maintained throughout all the culture stages leading to plant regeneration. More than 2500 explants from 1.5 to 3.0 cm inflorescences were bombarded and subjected to bialaphos selection. Out of more than 190 regenerated plants, 5 were determined to be Ignite resistant. Southern analyses confirmed the likelihood that the 5 herbicide resistant plants derived from two independent transformation events. The phosphinothricin acetyltransferase gene (bar) was inherited by and functionally expressed in T1 progeny. However, no β-glucuronidase (GUS) activity could be detected in T1 plants that contained uidA restriction fragments. Histological analyses indicated that in the absence of bialaphos morphogenesis was primarily via embryogenesis while organogenesis was more predominant in callus maintained with herbicide selection.

Characterization and in situ localization of a salt-induced tomato peroxidase mRNA.

NaCl treatment of tomato plants in hydroponic culture at concentrations as low as 50 mM resulted in enhanced accumulation of transcripts of TPX1, a full-length cDNA clone that we had isolated from a library of NaCl-treated tomato plants using a peroxidase-specific oligonucleotide probe. Although the overall amino acid sequence identity of TPX1 to other peroxidase genes was less than 45%, there was a very high degree of identity in all of the conserved domains. The deduced amino acid sequence included the presence of a N-terminal signal peptide but not the C-terminal extension present in peroxidases targeted to the vacuole. The mature protein has a theoretical pI value of 7.5. Transcripts that hybridized to TPX1 were detected only in the roots with higher levels of mRNA in epidermal and subepidermal cell layers. Isoelectric focusing of root extracts showed two major bands of peroxidase activity at pI 5.9 and 6.2. Both activities increased with salt treatment. Southern analysis indicated the presence of only a single TPX1 gene in tomato.

Asexual embryogenesis via callus of Theobroma cacao L.

Summary Callus of Theobroma cacao L. (cacao) possessing embryogenic competence occurred spontaneously with two clones of asexual embryos proliferated in vitro in a hormone-free basal medium by hypocotylary budding. Asexual embryogenesis via callus occurred at low frequency in the hormone-free basal medium. High concentrations of 2,4-D plus coconut water stimulated callus production and suppressed embryo production. Maximum frequency and intensity of embryogenesis occurred at 10−3 to 10−2 mg·l 2,4-D. Embryos originated from meristematic tissue at the periphery of callus clumps. During development asexual embryos either remained embedded in the callus or were connected through suspensor-like structures.

Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex

SummaryActivities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells ofZea mays — a species in which endomitosis occurs — andTulipa kaufmanniana — in which this process does not occur. InTulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. InZea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with3H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone.3H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments inZea mays and decreases slightly inTulipa kaufmanniana.It is argued that the differences between the incorporation of3H uridine and that or3H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.

Structure, Regulation and Function of the Osmotin Gene

Over the past years several genes have been reported to be osmotically regulated (Storey and Storey, 1981; Holtum and Winter, 1982; Singh et al., 1985; Singh et al., 1987a;Bedford et al., 1987; Close et al., 1989; Cushman et al., 1989; Singh et al., 1989a; 1989b; Delauney and Verma, 1990; Perez-Prat et al., 1990; Skriver and Mundy, 1990; Bartels et al., 1991; Dhindsa, 1991, Estragarcia et al., 1991; Narasimhan et al., 1991; Perez-Prat et al., 1992; Kononowicz et al., 1992; Nelson et al., 1992; Niu et al., 1993; Zhu et al., 1993b). These studies have been rationalized on the assumption that amongst these genes are molecular determinants of osmotic tolerance. Although the products of many of these genes still remain unidentified there are a number that have been well characterized and are of interest of several laboratories. One of these genes is osmotin (Singh et al., 1987a; LaRosa et al., 1989, Meeks-Wagner et al., 1989; Grosset et al., 1990; Roberts and Selitrennikoff, 1990; Stintzi et al., 1991; Woloshuk et al., 1991; Casas et al., 1992; LaRosa et al., 1992, Kononowicz et al., 1993).

Cell cycle duration in tobacco cells adapted to NaCl

Abstract Duration of the mitotic cycle phases of 428 mM NaCl-adapted and -unadapted cells was determined by the 3 H-thymidine “pulse-chase” technique and autoradiography. The cell cycle duration was estimated to be 22 hr for unadapted and 31 hr for adapted cells. Although all phases of the cell cycle were apparently prolonged in the adapted cells, most of the increase in the duration of the cell cycle was due to lengthening of the G 1 phase (from 9.7 to 16.6 hr) by ca 71%. Durations of the G 2 , S and M phases were extended by 14.5, 23.8 and 16.7%, respectively. Salt-adapted cells have a significantly increased cytoplasmic ion (Na + and Cl − ) content, reduced cell expansion and fresh weight gain as well as reduced RNA and protein syntheses, all of which may affect G 1 -attributed mechanisms that are responsible for progression through the cell cycle.

In situ 3H rRNA/DNA hybridization and silver staining of NORs during growth and differentiation of root cortex cells in the presence or absence of DNA endoreplication

The purpose of this study was to investigate changes in the number of NoRs in successive zones (1st, 3rd, 5th and 7th mm segments from the root tip) of growth and differentiation of roots in 4 plant species characterized by the presence or absence of endopolyploid nuclei. Cytophotometric measurements of the DNA content (after Feulgen’s procedure, hydrolysis for 1 h in 4N HC1 at 20 °C) have shown that in supra-meristematic zones (3rd, 5th and 7th segments) in Haemanthus katharinae and Tulipa kaufmanniana all nuclei have 2C DNA content (Figs. 3A and 4A), in Zea mays 4C and 8C nuclei are present, while in Helianthus annuus in the 5th and 7th segments some nuclei reveal 4C and 8C DNA content (comp. [8]).