Induction of a Pea Cell-Wall Invertase Gene by Wounding and Its Localized Expression in Phloem.

A full-length cell-wall invertase cDNA obtained from pea (Pisum sativum L.) seedlings was cloned previously. The gene exhibits tissue-specific expression, and both its transcript and enzyme activities are abundant only in root tissues. Mechanical wounding dramatically induced the accumulation of the cell-wall invertase mRNA in detached or intact leaves, stems, and roots. In both detached and intact tissues mRNA started to accumulate 3 h after wounding and in detached tissues (except root tissues) was much stronger and lasted longer compared with that in intact pea plants. The induction of cell-wall invertase by wounding was not systemic, since no significant increase of transcript was found in the unwounded tissues remote from the site of wounding. Accumulation of this cell-wall invertase was induced by abscisic or jasmonic acid, and in situ hybridization studies show that this invertase mRNA is differentially localized in wounded plant tissue, being most abundant in the phloem. mRNA accumulation was limited mainly to the wounded area; no significant increase was detected in the unwounded portions of the wounded stem segments. The results suggest that, as part of the wounding response, this cell-wall invertase may provide energy through hexose availability to companion cells in the phloem.

Gibberellin (GA3) enhances cell wall invertase activity and mRNA levels in elongating dwarf pea (Pisum sativum) shoots.

The invertase (EC 3.2.1.26) purified from cell walls of dwarf pea stems to homogeneity has a molecular mass of 64 kilodaltons (kD). Poly(A)+RNA was isolated from shoots of dwarf pea plants, and a cDNA library was constructed using lambda gt11 as an expression vector. The expression cDNA library was screened with polyclonal antibodies against pea cell wall invertase. One invertase cDNA clone was characterized as a full-length cDNA with 1,863 base pairs. Compared with other known invertases, one homologous region in the amino acid sequence was found. The conserved motif, Asn-Asp-Pro-Asn-Gly, is located near the N-terminal end of invertase. Northern blot analysis showed that the amount of invertase mRNA (1.86 kb) was rapidly induced to a maximal level 4 h after GA3 treatment, then gradually decreased to the control level. The mRNA level at 4 h in GA3-treated peas was fivefold higher than that of the control group. The maximal increase in activity of pea cell wall invertase elicited by GA3 occcured at 8 h after GA3 treatment. This invertase isoform was shown immunocytochemically to be localized in the cell walls, where a 10-fold higher accumulation occurred in GA3-treated tissue compared with control tissue. This study indicates that the expression of the pea shoot cell-wall invertase gene could be regulated by GA3 at transcriptional and/or translational levels.

Immunocytochemical localization of proteins in differentiating tissues of Pisum sativum.

Although there may be documented morphological changes during development, it is obvious that important changes in protein content occur in a vascular plant during the several stages of differentiation. In the absence of the latter information, an approach has been established for the localization of antigenic proteins in developing tissues of Pisum sativum. Monoclonal antibodies were raised to proteins extracted from pea internode tissue, and employed for the localization of three proteins in tissue sections. One of the proteins has two polypeptide subunits with molecular weights of 25,000 and 40,000 daltons, and the antibody binds to both of them. The three monoclonal antibodies produce different patterns of cellular localization in the tissue sections, as visualized by indirect immunocytochemical labeling. In another series of analyses, quantitative and qualitative differences in the protein contents of apical shoot tissue and mature internode shoot tissue have been found. These studies were based on the use of Western blots with both polyclonal (rabbit) antibodies and monoclonal (mouse) antibodies.

Nuclear and nucleolar protein during the cell cycle in differentiating Pisum sativum vascular tissue.

Squash preparations of Pisum sativum fourth internode tissue were stained with a combined Feulgen and dinitrofluorobenzene (DNFB) procedure. Nuclei from differentiating xylem vessel elements, phloem sieve tube elements and phloem fibres were measured for their DNA and protein contents with a Zeiss scanning cytophotometer linked to an interactive computer system. Nuclei were examined from both slowly growing and more rapidly growing internodes. A computer program was constructed to calculate nuclear protein alone as well as the ratio of DNFB (protein) to Feulgen (DNA) staining in each 0.5 X 0.5 micron measuring point. Nuclei were assigned to each of ten interphase fractions based on DNA content, nuclear area and percent condensed chromatin. There was a slight increase of nuclear protein during G1, a gradual increase in S, and a continued, often sharper, rise as G2 proceeded. In all three cell types, there was, on the average, a higher protein content throughout interphase in nuclei from the more rapidly growing internodes than from the slower growing ones. A population of fibre nuclei designated G0, however, differed from phloem and xylem G0 nuclei in the pattern of protein change. The nucleolar protein/DNA ratios of xylem nuclei increased in G1, showed no significant change in S, but increased thereafter.

Quantitative changes in the degree of chromatin condensation during the cell cycle in differentiating Pisum sativum vascular tissue.

Fixed tissue from the base of the fourth internode above the cotyledons of Pisum sativum plants were Feulgen-stained for DNA. In squash preparations three cell types: xylem vessel elements, phloem fibres and phloem sieve tube elements were identified. Measurement of Feulgen absorbance values in 0.5 X 0.5 microns measuring points across each nucleus were obtained with a scanning cytophotometer. Condensed chromatin was defined as any measuring point having an absorbance value greater than 0.32. Calculating 'percent condensed chromatin' as the percentage of the total nuclear DNA contained in those densely stained points, the pattern of change in this parameter during interphase in each cell type was observed. A generally similar pattern occurs in all three cell types. Percent condensed chromatin decreased from about 45% to 12% during G1, increased rapidly to the end of S, decreased during G2 and the following G1 to increase again during the subsequent S phase.

Protein staining methods in quantitative cytochemistry.

The chemical action and practical application of the Naphthol Yellow S, Alkaline Fast Green, Coomassie Brilliant Blue, Dinitrofluorobenzene and some lesser known protein staining methods have been surveyed with respect to their potentialities for quantitative cytochemical analyses. None of the dyes can be said to bind to any specific protein or group of proteins, but each may be used to analyse the presence of one or more particular amino acid residues. For the cytophotometric measurement of the 'total protein content' of individual cells and cell organelles the covalent binding Dinitrofluorobenzene and the electrostatic binding Naphthol Yellow S can properly be used. Fast Green FCF, applied at alkaline pH, binds electrostatically to the basic amino acid side chains of strongly basic proteins only but not in a quantitative (stoichiometrical) way. Coomassie Brilliant Blue, recently introduced to protein cytochemistry, may be useful for quantitative purposes. The combined Feulgen-Pararosaniline(SO2)/Naphthol Yellow S and Dinitrofluorobenzene/Feulgen-Pararosaniline(SO2) methods enable the simultaneous cytophotometric analysis at two different wavelengths for protein and DNA within the same microscopical preparation.

Constancy of DNA content in adrenal medulla nuclei of cold-treated rats.

Reports of changes in DNA content of certain types of cells following exposure to conditions of stress has led to the suggestion that two kinds of DNA may be present. One is genetic DNA, and the other is called "metabolic" DNA. In a further attempt to investigate the possibility of this phenomenon, determinations of DNA content were made on Feulgen-stained nuclei of adrenal glands and kidneys in cold-treated rats. Feulgen-stained nuclei were measured by two-wavelength microspectrophotometry. Particular attention was given to the handling of the smears in hydrolysis and staining. Mean values of Feulgen-DNA contents in a total of 720 nuclei demonstrated (a) a constancy of DNA content within 2% in individual nuclei both in adrenal medulla and kidney cortex, (b) no more than an average of 2% difference in DNA content between control and experimental nuclei, and (c) no more than an average of 1.5% difference in DNA content between normal kidney cortex nuclei and normal adrenal medulla nuclei. These results confirm the view that the more precise the measurement, the more accurately the constancy rule is obeyed. Moreover, there is no support for the concept of a metabolic DNA in the rat adrenal medulla.