Badia Bisbis

Concepts in plant stress physiology. Application to plant tissue cultures

Because the term stress is used, most often subjectively, with variousmeanings, this paper first attempts to clarify the physiological definition,andthe appropriate terms as responses in different situations. The flexibility ofnormal metabolism allows the development of responses to environmental changeswhich fluctuate regularly and predictably over daily and seasonal cycles. Thusevery deviation of a factor from its optimum does not necessarily result instress. Stress begins with a constraint or with highly unpredictablefluctuations imposed on regular metabolic patterns that cause bodily injury,disease, or aberrant physiology. Stress is the altered physiological conditioncaused by factors that tend to alter an equilibrium. Strain is any physicaland/or chemical change produced by a stress, i.e. every established condition,which forces a system away from its thermodynamic optimal state. The papersecondly summarises the Strassers state-change concept which is preciselythat suboptimality is the driving force for acclimation (genotype level) oradaptation (population level) to stress. The paper continues with the actualknowledge on the mechanisms of stress recognition and cell signalling. Briefly:plasma membranes are the sensors of environmental changes; phytohormones andsecond messengers are the transducers of information from membranes tometabolism; carbon balance is the master integrator of plant response; betwixtand between, some genes are expressed more strongly, whereas others arerepressed. Reactive oxygen species play key roles in up- and down-regulation ofmetabolism and structure. The paper shows finally that the above concepts canbeapplied to plant tissue cultures where the accumulating physiological andgenetical deviations (from a normal plant behaviour) are related to thestressing conditions of the in vitro culture media and ofthe confined environment. The hyperhydrated state of shoots and the cancerousstate of cells, both induced under conditions of stress in invitro cultures, are identified and detailed, because they perfectlyillustrate the stress-induced state-change concept. It is concluded that stressresponses include either pathologies or adaptive advantages. Stress may thuscontain both destructive and constructive elements : it is a selection factoraswell as a driving force for improved resistance and adaptive evolution.

Loss of plant organogenic totipotency in the course of in vitro neoplastic progression

SummaryThe aptitude for organogenesis from normal hormone-dependent cultures very commonly decreases as the tissues are serially subcultured. The reasons for the loss of regenerative ability may vary under different circumstances: genetic variation in the cell population, epigenetic changes, disappearance of an organogenesis-promoting substance, etc. The same reasons may be evoked for the progressive and eventually irreversible loss of organogenic totipotency in the course of neoplastic progressions from hormone-independent tumors and hyperhydric teratomas to cancers. As in animal cells, plant cells at the end of a neoplastic progression have probably undergone several independent genetic accidents with cumulative effects. They indeed are characterized by atypical biochemical cycles from which they are apparently unable to escape. The metabolic changes are probably not the primary defects that cause cancer, rather they may allow the cells to survive. How these changes, namely an oxidative stress, affect organogenesis is not known. The literature focuses on somatic mutations and epigenetic changes that cause aberrant regulation of cell cycle genes and their machinery.

Atypical metabolisms and biochemical cycles imposing the cancerous state on plant cells

The biological, morphological and biochemical characteristics which define plant cancer cells at the end of a neoplasic progression in the absence of pathogens and which distinguish them from tumorous cells are summarized. Such plant cancer cells have in common with animal cancer cells many metabolic disturbances. The present paper reviews the biochemical changes in nitrogen, carbon, sugar and heme metabolisms which contribute to polyamine (PAs) accumulation. It indicates how these changes are interconnected and even form between each other biochemical cycles which likely maintain these cells in their irreversible state. The role of these cycles in the maintenance of such cells under a probable permanent oxidative stress is debated.

Restart of Lignification in Micropropagated Walnut Shoots Coincides with Rooting Induction

The lignin content of walnut shoots did not change during in vitro shoot multiplication. Lignin content started to increase as soon as shoots were passed to a rooting medium with auxin. Exogenous auxin (applied for rooting) caused a transient elevation of the endogenous free indoleacetic acid (IAA) content with a simultaneous decrease of peroxidase activity. These events typically marked the completion of the rooting inductive phase (before any visible histological event, that is before the cell divisions beginning the rooting initiation phase). This meant that either the given exogenous auxin or the endogenous IAA has served as signal for the stimulation of lignification. Continued increase of lignification in the shoots required completion of root formation; this increase indeed was slown down when root emergence did not occur. It was further shown that lignification varied conversely to the content of the soluble phenol content, itself apparently being related to the activity of phenylalanine ammonia-lyase activity.

Darkness improves growth and delays necrosis in a nonchlorophyllous habituated sugarbeet callus: Biochemical changes

The transfer of light-cultured green normal (N) and white habituated (HNO) sugarbeet callus to darkness reduced the growth of N callus and improved growth and delayed necrosis in the HNO callus. The decrease of dry matter of N callus under darkness was accompanied by a reduced content of carotenoids and by decreased CO2 fixation, which was compensated by an increased dependency on externally supplied sucrose. The levels of some organic nitrogen compounds such as glutamate, proline, and free polyamines were not affected by transfer to darkness of N or HNO callus. Darkness decreased ethylene emissions in both callus types. In the HNO callus, the sucrose growth dependency and the CO2 fixation were unaffected by darkness. Chlorophylls were absent both in light and darkness, whereas some carotenoids were accumulated in the HNO callus only in dark conditions. In another connection, a significant increase of peroxidase activity, which did not occur in the N callus, was induced by darkness in the HNO callus. A decreased content of thio-barbituric acid (TBA)-reactive substances was measured in the HNO callus transferred to darkness, whereas an increase was noticed in the N callus placed in the same conditions. These metabolic changes and the reduction of cellular damage in darkness revealed light-induced stress reactions leading to necrosis and to reduced growth of HNO callus. It appeared that darkness allowed the HNO callus to avoid the photooxidation stress. Therefore, the favorable effect of darkness on HNO growth might be explained by the suppression of photooxidative damage due to the absence of carotenoids. The higher peroxidase activity in the HNO callus maintained in darkness raised the problem of heme synthesis in this heterotrophic callus.

Chlorophylls and carotenoids in a fully habituated nonorganogenic callus of Beta vulgaris

A fully habituated (H) nonorganogenic sugar beet callus, subcultured in the light, did not contain detectable chlorophyll (Chl) nor carotenoid (Car). It accumulated some Car in the dark. Fluorescence spectra indicated that this H callus also accumulated some protochlorophyllide which, however, was not well integrated into the protochlorophyllide-NADPH-photoreductase complex, and therefore not transformed into chlorophyllide in the light. The H callus showed no variable fluorescence which indicated absence of photosynthesis, and therefore it suggested a full heterotrophic behaviour of this peculiar callus line. A green hormone-dependent callus of the same sugar beet had normal fluorescence spectra and kinetics comparable to those of a green leaf.

Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris (L.)

Habituated (H) nonorganogenic sugarbeet callus was found to exhibit a disturbed sugar metabolism. In contrast to cells from normal (N) callus, H cells accumulate glucose and fructose and show an abnormal high fructose/glucose ratio. Moreover, H cells which have decreased wall components, display lower glycolytic enzyme activities (hexose phosphate isomerase and phosphofructokinase) which is compensated by higher activities of the enzymes of the hexose monophosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). The disturbed sugar metabolism of the H callus is discussed in relation to a deficiency in H2O2 detoxifying systems.

Flow Cytometry Estimation of Nuclear Size and Ploidy Level of Habituated Calli of Sugar Beet

A fully habituated (auxin- and cytokinin-independent) self-regenerating (organo-genic) sugar beet cell line (HO) and a fully habituated non-organogenic one (HNO) derived from the former one, were analyzed as to their nuclear size and DNA content. Flow cytometry and image analysis were used and cells of certified diploid leaves of the same sugar beet strain served as controls. The HNO cells had been shown previously to have many characteristics of cancerous cells. The analyses made on leaves and HNO cells indicated the presence of only one population of cycling cells. In HO cells, two cycling populations were detected: the first one had the same DNA content as the leaves while the second one contained two fold more DNA than the first population. HNO cells showed the higher nuclear size and DNA content. HNO cells also showed evidence of aneuploidy. Thus, nuclear size, DNA content and ploidy level increase together with the neoplasic progression to culminate in HNO cells with the loss of organogenic totipotency.

Putrescine Metabolism in a Fully Habituated Nonorganogenic Sugar Beet Callus and its Relationship with Growth

Summary A fully-habituated and nonorganogenic (HNO) sugar beet callus was previously shown to overproduce polyamines, as compared with a normal (N) auxin- and cytokinin-dependent callus of the same strain. Because relationships were established between polyamine levels and metabolism with different growth and development processes, some key enzymes in the metabolic pathways of polyamines were investigated in the HNO callus, and their involvement in growth appraised. Putrescine was found to be the major free and conjugated polyamine in the HNO callus. It was biosynthesised preferentially via ornithine and ornithine decarboxylase (ODC), which is in agreement with the surplus of synthesised ornithine. Diamine (DAO) and polyamine (PAO)-oxidase activities were also highest in the HNO callus, as compared with the normal, with DAO being the more active. Transglutaminase activities(± Ca) were also higher in HNO than in normal callus. Addition of different polyamines or of inhibitors of their biosynthesis to the culture medium of the HNO callus modified the level of endogenous polyamines and affected callus growth. The results thus pointed out a higher polyamine metabolism, particularly of putrescine, in the actively growing auxin- and cytokinin-independent callus than in the normal one. They also provided evidence for the sensitivity of a habituated tissue type towards this class of growth regulators.

Biosynthesis of 5-aminolevulinic Acid via the Shemin Pathway in a Green Sugar Beet Callus

Abstract5-Aminolevulinic acid synthase (ALAS) has been detected in a normal (auxin- and cytokinin-dependent) green sugar beet callus under light and under darkness. ALAS activity was lower when the callus was grown under light. The supply of precursors of the Shemin pathway (glycine and succinate) to dark-grown callus enhanced considerably the capacity of the 5-aminolevulinic acid (ALA) formation. Glutamate, γ-aminobutyrate or α-ketoglutarate also increased ALA accumulation. Such an accumulation was also obtained after inhibition of polyamine synthesis. The results show that glutamate or its derivatives might feed the Shemin pathway in conditions preventing glutamate to be used through the Beale pathway.