Nitric oxide alleviates cadmium- but not arsenic-induced damages in rice roots.

Nitric oxide (NO) has signalling roles in plant stress responses. Cadmium (Cd) and arsenic (As) soil pollutants alter plant development, mainly the root-system, by increasing NO-content, triggering reactive oxygen species (ROS), and forming peroxynitrite by NO-reaction with the superoxide anion. Interactions of NO with ROS and peroxynitrite seem important for plant tolerance to heavy metal(oid)s, but the mechanisms underlying this process remain unclear. Our goal was to investigate NO-involvement in rice (Oryza sativa L.) root-system after exposure to Cd or As, to highlight possible differences in NO-behaviour between the two pollutants. To the aim, morpho-histological, chemical and epifluorescence analyses were carried out on roots of different origin in the root-system, under exposure to Cd or As, combined or not with sodium nitroprusside (SNP), a NO-donor compound. Results show that increased intracellular NO levels alleviate the root-system alterations induced by Cd, i.e., inhibition of adventitious root elongation and lateral root formation, increment in lignin deposition in the sclerenchyma/endodermis cell-walls, but, even if reducing As-induced endodermis lignification, do not recover the majority of the As-damages, i.e., enhancement of AR-elongation, reduction of LR-formation, anomalous tissue-proliferation. However, NO decreases both Cd and As uptake, without affecting the pollutants translocation-capability from roots to shoots. Moreover, NO reduces the Cd-induced, but not the As-induced, ROS levels by triggering peroxynitrite production. Altogether, results highlight a different behaviour of NO in modulating rice root-system response to the toxicity of the heavy metal Cd and the metalloid As, which depends by the NO-interaction with the specific pollutant.

Cadmium and arsenic-induced-stress differentially modulates Arabidopsis root architecture, peroxisome distribution, enzymatic activities and their nitric oxide content.

In plant cells, cadmium (Cd) and arsenic (As) exert toxicity mainly by inducing oxidative stress through an imbalance between the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and their detoxification. Nitric oxide (NO) is a RNS acting as signalling molecule coordinating plant development and stress responses, but also as oxidative stress inducer, depending on its cellular concentration. Peroxisomes are versatile organelles involved in plant metabolism and signalling, with a role in cellular redox balance thanks to their antioxidant enzymes, and their RNS (mainly NO) and ROS. This study analysed Cd or As effects on peroxisomes, and NO production and distribution in the root system, including primary root (PR) and lateral roots (LRs). Arabidopsis thaliana wild-type and transgenic plants enabling peroxisomes to be visualized in vivo, through the expression of the 35S-cyan fluorescent protein fused to the peroxisomal targeting signal1 (PTS1) were used. Peroxisomal enzymatic activities including the antioxidant catalase, the H2O2-generating glycolate oxidase, and the hydroxypyruvate reductase, and root system morphology were also evaluated under Cd/As exposure. Results showed that Cd and As differently modulate these activities, however, catalase activity was inhibited by both. Moreover, Arabidopsis root system was altered, with the pollutants differently affecting PR growth, but similarly enhancing LR formation. Only in the PR apex, and not in LR one, Cd more than As caused significant changes in peroxisome distribution, size, and in peroxisomal NO content. By contrast, neither pollutant caused significant changes in peroxisomes size and peroxisomal NO content in the LR apex.

Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into indole-3-acetic acid and stimulation of anthranilate synthase activity.

BACKGROUND: Indole-3-acetic acid (IAA), and its precursor indole-3-butyric acid (IBA), control adventitious root (AR) formation in planta. Adventitious roots are also crucial for propagation via cuttings. However, IBA role(s) is/are still far to be elucidated. In Arabidopsis thaliana stem cuttings, 10 µM IBA is more AR-inductive than 10 µM IAA, and, in thin cell layers (TCLs), IBA induces ARs when combined with 0.1 µM kinetin (Kin). It is unknown whether arabidopsis TCLs produce ARs under IBA alone (10 µM) or IAA alone (10 µM), and whether they contain endogenous IAA/IBA at culture onset, possibly interfering with the exogenous IBA/IAA input. Moreover, it is unknown whether an IBA-to-IAA conversion is active in TCLs, and positively affects AR formation, possibly through the activity of the nitric oxide (NO) deriving from the conversion process. RESULTS: Revealed undetectable levels of both auxins at culture onset, showing that arabidopsis TCLs were optimal for investigating AR-formation under the total control of exogenous auxins. The AR-response of TCLs from various ecotypes, transgenic lines and knockout mutants was analyzed under different treatments. It was shown that ARs are better induced by IBA than IAA and IBA + Kin. IBA induced IAA-efflux (PIN1) and IAA-influx (AUX1/LAX3) genes, IAA-influx carriers activities, and expression of ANTHRANILATE SYNTHASE -alpha1 (ASA1), a gene involved in IAA-biosynthesis. ASA1 and ANTHRANILATE SYNTHASE -beta1 (ASB1), the other subunit of the same enzyme, positively affected AR-formation in the presence of exogenous IBA, because the AR-response in the TCLs of their mutant wei2wei7 was highly reduced. The AR-response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA-conversion, was also strongly reduced. Nitric oxide, an IAA downstream signal and a by-product of IBA-to-IAA conversion, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter explants. CONCLUSIONS: Altogether, results showed that IBA induced AR-formation by conversion into IAA involving NO activity, and by a positive action on IAA-transport and ASA1/ASB1-mediated IAA-biosynthesis. Results are important for applications aimed to overcome rooting recalcitrance in species of economic value, but mainly for helping to understand IBA involvement in the natural process of adventitious rooting.

Ethylene and auxin interaction in the control of adventitious rooting in Arabidopsis thaliana.

Adventitious roots (ARs) are post-embryonic roots essential for plant survival and propagation. Indole-3-acetic acid (IAA) is the auxin that controls AR formation; however, its precursor indole-3-butyric acid (IBA) is known to enhance it. Ethylene affects many auxin-dependent processes by affecting IAA synthesis, transport and/or signaling, but its role in AR formation has not been elucidated. This research investigated the role of ethylene in AR formation in dark-grown Arabidopsis thaliana seedlings, and its interaction with IAA/IBA. A number of mutants/transgenic lines were exposed to various treatments, and mRNA in situ hybridizations were carried out and hormones were quantified In the wild-type, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) at 0.1 µM enhanced AR formation when combined with IBA (10 µM), but reduced it when applied alone; this effect did not occur in the ein3eil1 ethylene-insensitive mutant. ACC inhibited the expression of the IAA-biosynthetic genes WEI2, WEI7, and YUC6, but enhanced IBA-to-IAA conversion, as shown by the response of the ech2ibr10 mutant and an increase in the endogenous levels of IAA. The ethylene effect was independent of auxin-signaling by TIR1-AFB2 and IBA-efflux by ABCG carriers, but it was dependent on IAA-influx by AUX1/LAX3.Taken together, the results demonstrate that a crosstalk involving ethylene signaling, IAA-influx, and IBA-to-IAA conversion exists between ethylene and IAA in the control of AR formation.

Arabidopsis SHR and SCR transcription factors and AUX1 auxin influx carrier control the switch between adventitious rooting and xylogenesis in planta and in in vitro cultured thin cell layers.

BACKGROUND AND AIMS: Adventitious roots (ARs) are essential for vegetative propagation. The Arabidopsis thaliana transcription factors SHORT ROOT (SHR) and SCARECROW (SCR) affect primary/lateral root development, but their involvement in AR formation is uncertain. LAX3 and AUX1 auxin influx carriers contribute to primary/lateral root development. LAX3 expression is regulated by SHR, and LAX3 contributes to AR tip auxin maximum. In contrast, AUX1 involvement in AR development is unknown. Xylogenesis is induced by auxin plus cytokinin as is AR formation, but the genes involved are largely unknown. Stem thin cell layers (TCLs) form ARs and undergo xylogenesis under the same auxin plus cytokinin input. The aim of this research was to investigate SHR, SCR, AUX1 and LAX3 involvement in AR formation and xylogenesis in intact hypocotyls and stem TCLs in arabidopsis. METHODS: Hypocotyls of scr-1, shr-1, lax3, aux1-21 and lax3/aux1-21 Arabidopsis thaliana null mutant seedlings grown with or without auxin plus cytokinin were examined histologically, as were stem TCLs cultured with auxin plus cytokinin. SCR and AUX1 expression was monitored using pSCR::GFP and AUX1::GUS lines, and LAX3 expression and auxin localization during xylogenesis were monitored by using LAX3::GUS and DR5::GUS lines. KEY RESULTS: AR formation was inhibited in all mutants, except lax3. SCR was expressed in pericycle anticlinally derived AR-forming cells of intact hypocotyls, and in cell clumps forming AR meristemoids of TCLs. The apex was anomalous in shr and scr ARs. In all mutant hypocotyls, the pericycle divided periclinally to produce xylogenesis. Xylary element maturation was favoured by auxin plus cytokinin in shr and aux1-21. Xylogenesis was enhanced in TCLs, and in aux1-21 and shr in particular. AUX1 was expressed before LAX3, i.e. in the early derivatives leading to either ARs or xylogenesis. CONCLUSIONS: AR formation and xylogenesis are developmental programmes that are inversely related, but they involve fine-tuning by the same proteins, namely SHR, SCR and AUX1. Pericycle activity is central for the equilibrium between xylary development and AR formation in the hypocotyl, with a role for AUX1 in switching between, and balancing of, the two developmental programmes.

Auxin and cytokinin control formation of the quiescent centre in the adventitious root apex of Arabidopsis.

BACKGROUND AND AIMS: Adventitious roots (ARs) are part of the root system in numerous plants, and are required for successful micropropagation. In the Arabidopsis thaliana primary root (PR) and lateral roots (LRs), the quiescent centre (QC) in the stem cell niche of the meristem controls apical growth with the involvement of auxin and cytokinin. In arabidopsis, ARs emerge in planta from the hypocotyl pericycle, and from different tissues in in vitro cultured explants, e.g. from the stem endodermis in thin cell layer (TCL) explants. The aim of this study was to investigate the establishment and maintenance of the QC in arabidopsis ARs, in planta and in TCL explants, because information about this process is still lacking, and it has potential use for biotechnological applications. METHODS: Expression of PR/LR QC markers and auxin influx (LAX3)/efflux (PIN1) genes was investigated in the presence/absence of exogenous auxin and cytokinin. Auxin was monitored by the DR5::GUS system and cytokinin by immunolocalization. The expression of the auxin-biosynthetic YUCCA6 gene was also investigated by in situ hybridization in planta and in AR-forming TCLs from the indole acetic acid (IAA)-overproducing superroot2-1 mutant and its wild type. KEY RESULTS: The accumulation of auxin and the expression of the QC marker WOX5 characterized the early derivatives of the AR founder cells, in planta and in in vitro cultured TCLs. By determination of PIN1 auxin efflux carrier and LAX3 auxin influx carrier activities, an auxin maximum was determined to occur at the AR tip, to which WOX5 expression was restricted, establishing the positioning of the QC. Cytokinin caused a restriction of LAX3 and PIN1 expression domains, and concomitantly the auxin biosynthesis YUCCA6 gene was expressed in the apex. CONCLUSIONS: In ARs formed in planta and TCLs, the QC is established in a similar way, and auxin transport and biosynthesis are involved through cytokinin tuning.

Cold perception and gene expression differ in Olea europaea seed coat and embryo during drupe cold acclimation.

FAD2 and FAD7 desaturases are involved in cold acclimation of olive (Olea europaea) mesocarp. There is no research information available on cold acclimation of seeds during mesocarp cold acclimation or on differences in the cold response of the seed coat and embryo. How FAD2 and FAD7 affect seed coat and embryo cold responses is unknown. Osmotin positively affects cold acclimation in olive tree vegetative organs, but its role in the seeds requires investigation. OeFAD2.1, OeFAD2.2, OeFAD7 and Oeosmotin were investigated before and after mesocarp acclimation by transcriptomic, lipidomic and immunolabelling analyses, and cytosolic calcium concentration ([Ca(2+)](cyt)) signalling, F-actin changes and seed development were investigated by epifluorescence/histological analyses. Transient [Ca(2+)](cyt) rises and F-actin disassembly were found in cold-shocked protoplasts from the seed coat, but not from the embryo. The thickness of the outer endosperm cuticle increased during drupe exposure to lowering of temperature, whereas the embryo protoderm always lacked cuticle. OeFAD2 transcription increased in both the embryo and seed coat in the cold-acclimated drupe, but linoleic acid (i.e. the product of FAD2 activity) increased solely in the seed coat. Osmotin was immunodetected in the seed coat and endosperm of the cold-acclimated drupe, and not in the embryo. The results show cold responsiveness in the seed coat and cold tolerance in the embryo. We propose a role for the seed coat in maintaining embryo cold tolerance by increasing endosperm cutinization through FAD2 and osmotin activities.

Two SERK genes are markers of pluripotency in Cyclamen persicum Mill.

The genetic basis of stem cell specification in somatic embryogenesis and organogenesis is still obscure. SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes are involved in embryogenesis and organogenesis in numerous species. In vitro culture of Cyclamen persicum immature ovules provides a system for investigating stem cell formation and maintenance, because lines forming either organs or embryos or callus without organs/embryos are available for the same cultivar and plant growth regulator conditions. The present aim was to exploit this property of cyclamen cultures to understand the role of SERK(s) in stem cell formation and maintenance in somatic embryogenesis and organogenesis in vitro, in comparison with expression in planta. CpSERK1 and CpSERK2 were isolated from embryogenic callus. CpSERK1 and CpSERK2 levels by RT-PCR showed that expression is high in embryogenic, moderate in organogenic, and null in recalcitrant calli. in situ hybridizations showed that the expression of both genes started in clumps of pluripotent stem cells, from which both pre-embryogenic aggregates and organ meristemoids derived, and continued in their trans-amplifying, meristem-like, derivatives. Expression declined in organ meristemoids, in parallel with a partial loss of meristematization. In mature somatic embryos, and in shoot and root primordia, CpSERK1 and CpSERK2 were expressed in meristems, and similar patterns occurred in zygotic embryo and primary meristems in planta. The results point to SERK1 and SERK2 as markers of pluripotency in cyclamen. It is proposed that the high expression of these genes in the trans-amplifying derivatives of the stem cells maintains a pluripotent condition leading to totipotency and, consequently, somatic embryogenesis.

Cold affects the transcription of fatty acid desaturases and oil quality in the fruit of Olea europaea L. genotypes with different cold hardiness.

The olive tree lacks dormancy and is low temperature sensitive, with differences in cold tolerance and oil quality among genotypes. The oil is produced in the drupe, and the unsaturated fatty acids contribute to its quality. The aim of the present research was to investigate the relationship among development, cold response, expression of fatty acid desaturase (FAD) genes, and unsaturated fatty acid composition in drupes belonging to genotypes differing in leaf cold tolerance, but producing good oil (i.e. the non-hardy Moraiolo, the semi-hardy Frantoio, and the hardy Canino). In all genotypes, cold sensitivity, evaluated by cold-induced transient increases in cytosolic calcium, was high in the epi-mesocarp cells before oil body formation, and decreased during oil biogenesis. However, genotype-dependent differences in cold sensitivity appeared at the end of oil production. Genotype-dependent differences in FAD2.1, FAD2.2, FAD6, and FAD7 expression levels occurred in the epi-mesocarp cells during the oleogenic period. However, FAD2.1 and FAD7 were always the highest in the first part of this period. FAD2.2 and FAD7 increased after cold applications during oleogenesis, independently of the genotype. Unsaturated fatty acids increased in the drupes of the non-hardy genotype, but not in those of the hardy one, after cold exposure at the time of the highest FAD transcription. The results show a direct relationship between FAD expression and lipid desaturation in the drupes of the cold-sensitive genotype, and an inverse relationship in those of the cold-resistant genotype, suggesting that drupe cold acclimation requires a fine FAD post-transcriptional regulation. Hypotheses relating FAD desaturation to storage and membrane lipids, and genotype cold hardiness are discussed.

Polyamine biosynthesis and control of the development of functional pollen in kiwifruit.

The role of polyamines (PAs) in plant reproduction, especially pollen development and germination has been demonstrated in several higher plants. The aim of the present research was to investigate PA involvement in pollen development and germination in dioecious kiwifruit (Actinidia deliciosa). Differences in PA content, level and gene expression for PA biosynthetic enzymes, and the effect of PA biosynthetic inhibitors were found during pollen development (or abortion in female flowers). Whereas PAs, especially spermidine (Spd), remained high throughout the development of functional pollen, the levels collapsed by the last stage of development of sterile pollen. Mature and functional pollen from male-fertile anthers showed S-adenosyl methionine decarboxylase activity (SAMDC; involved in Spd biosynthesis) throughout microgametogenesis, with high levels of soluble SAMDC found starting from the late uninucleate microspore stage. Soluble SAMDC was absent in male-sterile anthers. Arginine decarboxylase [ADC; for putrescine (Put) biosynthesis] showed little difference in functional vs sterile pollen; ornithine decarboxylase [ODC; also for putrescine (Put) biosynthesis] was present only in sterile pollen. Ultrastructural studies of aborted pollen grains in male-sterile flowers showed that cytoplasmic residues near the intine contain vesicles, extruding towards the pollen wall. Very high SAMDC activity was found in the wall residues of the aborted pollen. The combined application in planta of competitive inhibitors of S-adenosylmethionine decarboxylase (MGBG) and of spermidine synthase (CHA), or of D-arginine (inhibitor of Put synthesis), to male-fertile plants led to abnormal pollen grains with reduced viability. The importance of PAs during male-fertile pollen germination was also found. In fact, PA biosynthetic enzymes (ADC and, mainly, SAMDC) were active early during pollen hydration and germination in vitro. Two different SAMDC gene transcripts were expressed in germinating pollen together with a lower level of ADC transcript. Gene expression preceded PA enzyme activity. The application of PA inhibitors in planta drastically reduced pollen germination. Thus, low free Spd can lead either to degeneration or loss of functionality of kiwifruit pollen grains.

Osmotin induces cold protection in olive trees by affecting programmed cell death and cytoskeleton organization.

Osmotin is a pathogenesis-related protein exhibiting cryoprotective functions. Our aim was to understand whether it is involved in the cold acclimation of the olive tree (Olea europaea L.), a frost-sensitive species lacking dormancy. We exposed olive trees expressing tobacco osmotin gene under the 35S promoter (35S:osm) [in the same manner as wild type (wt) plants] to cold shocks in the presence/absence of cold acclimation, and monitored changes in programmed cell death (PCD), cytoskeleton, and calcium ([Ca2+]c) signalling. In the wt, osmotin was immunolocalized only in cold-acclimated plants, and in the tissues showing PCD. In the 35S:osm clones, the protein was detected also in the non-acclimated plants, and always in the tissues exhibiting PCD. In the non-acclimated wt protoplasts exposed to cold shock, a transient decrease in phallotoxin signal suggests a temporary disassembly of F-actin, a transient increase occurred instead in 35S:osm protoplasts exposed to the same shock. Transient increases in [Ca2+]c were observed only in the wt protoplasts. However, when F-actin was depolymerized by cytochalasin or latrunculin, and microtubules by colchicine, increase in [Ca2+]c also occurred in the 35S:osm protoplasts. Successive cold shocks caused transient rises in [Ca2+]c and transient decreases in the phallotoxin signal in wt protoplasts. No change occurred in [Ca2+]c occurred in the 35S:osm protoplasts. The phallotoxin signal transiently increased at the first shock, but did not change after the subsequent shocks, and an overall signal reduction occurred with shock repetition. Following acclimation, no cold shock-induced change in [Ca2+]c levels and F-actin signal occurred either in wt or 35S:osm protoplasts. The results show that osmotin is positively involved in the acclimation-related PCD, in blocking the cold-induced calcium signalling, and in affecting cytoskeleton in response to cold stimuli.

Methyl jasmonate disrupts shoot formation in tobacco thin cell layers by over-inducing mitotic activity and cell expansion.

The aim of the present study was to determine early cyto-histological events associated with the reduced number of shoots formed at the end of culture in tobacco (Nicotiana tabacum L.) thin cell layers treated with methyl jasmonate (MJ) [S. Biondi et al. (2001) J Exp Bot 52:1-12]. The results show that 0.1-10 microM MJ strongly stimulated mitotic activity early in culture relative to untreated controls. Treatment with MJ also induced anomalous mitoses. Enhanced proliferative growth was confirmed by northern analysis and in situ hybridisation using cDNA probes of the G1/S phase-specific genes ubiquitin carboxyl-extension protein (ubi-CEP), topoisomerase 1 (top1) and ribonucleotide reductase (RNR). The formation of meristematic cell clusters on day 5 was also enhanced by 1 muM MJ, but subsequent development of these cell clusters into meristemoids and shoot primordia was reduced by all MJ concentrations in a dose-dependent manner. Cell expansion was stimulated by MJ concentrations ranging from 0.001 to 10 microM; expanded cells frequently occurred around and within meristemoids and shoot primordia, and displayed thickened and suberised cell walls; cell wall thickness increased with increasing MJ concentration. These cytological events caused alterations in the tunica and stem differentiation of the shoot dome. The apparently paradoxical role of MJ, which deregulates shoot formation through a stimulation of growth events, i.e., mitotic activity and cell expansion, is discussed.

Adventitious root formation in Arabidopsis thaliana thin cell layers.

This paper describes, for the first time, de novo adventitious root formation from thin cell layers (TCLs) of Arabidopsis thaliana. The objective of the study was to determine the optimal hormonal and light conditions and the optimal exogenous Ca2+ concentration for obtaining adventitious rooting (AR) from A. thaliana TCLs and to identify the tissue(s) involved in the process. The results show that maximum AR was obtained with a single-phase method in the presence of 10 microM indole-3-butyric acid and 0.1 microM kinetin under continuous darkness for 30 days and with 0.6 mM exogenous CaCl2. The endodermis was the only tissue involved in root meristemoid formation. The role of Ca2+ in AR and the importance of using Arabidopsis TCLs in studies on the genetic/biochemical control of AR are discussed.

The arabidopsis ATHB-8 HD-zip protein acts as a differentiation-promoting transcription factor of the vascular meristems.

ATHB-8, -9, -14, -15, and IFL1/REV are members of a small homeodomain-leucine zipper family whose genes are characterized by expression in the vascular tissue. ATHB-8, a gene positively regulated by auxin (Baima et al., 1995), is considered an early marker of the procambial cells and of the cambium during vascular regeneration after wounding. Here, we demonstrate that although the formation of the vascular system is not affected in athb8 mutants, ectopic expression of ATHB-8 in Arabidopsis plants increased the production of xylem tissue. In particular, a careful anatomical analysis of the transgenic plants indicated that the overexpression of ATHB-8 promotes vascular cell differentiation. First, the procambial cells differentiated precociously into primary xylem. In addition, interfascicular cells also differentiated precociously into fibers. Finally, the transition to secondary growth, mainly producing xylem, was anticipated in transgenic inflorescence stems compared with controls. The stimulation of primary and secondary vascular cell differentiation resulted in complex modifications of the growth and development of the ATHB-8 transgenic plants. Taken together, these results are consistent with the hypothesis that ATHB-8 is a positive regulator of proliferation and differentiation, and participates in a positive feedback loop in which auxin signaling induces the expression of ATHB-8, which in turn positively modulates the activity of procambial and cambial cells to differentiate.

Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers.

The effect of methyl jasmonate (MJ) on de novo shoot formation and polyamine metabolism was investigated in thin layer explants of tobacco (Nicotiana tabacum L. cv. Samsun). A relatively low concentration of MJ (0.1 microM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 microM) significantly inhibited organogenesis. The histological study revealed that, with increasing concentrations of MJ, the formation of meristemoids and shoot domes declined and the incidence of cell hypertrophy increased. In explants cultured with 0.1, 1 or 10 microM MJ, the endogenous levels of free putrescine, spermidine and spermine generally declined compared with controls, after 7 and 15 d. Perchloric acid-soluble conjugated polyamines accumulated dramatically during culture, but much more so in the presence of MJ than in controls. Acid-insoluble conjugated spermidine alone increased in response to the elicitor. Activities of the putrescine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) in the soluble fraction of MJ-treated explants displayed up to 3-fold increases relative to control explants. However, the most relevant increases in these enzyme activities occurred in the particulate fraction. The activity of S:-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis, was also stimulated by exposure to MJ. Northern analyses revealed MJ-induced, generally dose-dependent, increases in the mRNA levels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6) activity was stimulated by MJ mainly in the cell wall fraction. The upregulation of polyamine metabolism is discussed in relation to the morphogenic behaviour of MJ-treated explants.

Early physiological and cytological events induced by wounding in potato tuber.

The response of potato tuber (Solanum tuberosum L. cv. Kennebec) to mechanical wounding was investigated at different times. Changes in the levels of indole-3-acetic acid (IAA), polyunsaturated fatty acids (PUFAs) and lipid hydroperoxides (LOOHs) were monitored up to 120 min after wounding and related to the cytological events occurring up to 24 h. Twenty minutes after injury, an increase in IAA and LOOH levels and a decrease in the levels of PUFAs was observed. Wounding induced mitoses in differentiated (parenchyma) cells starting at 120 min, and promoted an increase of mitotic activity in the meristematic cells (procambium and bud dome), after 360 min. The inhibition of the increase in LOOHs and IAA by lipoxygenase (LOX) inhibitors, as well as the ability of in vitro peroxidated linoleic acid to enhance IAA production, suggest a close relationship among lipoperoxidation, IAA and mitotic activity in the response of potato tuber cells to injury, resulting in a specific growth response, i.e. bud growth and periderm formation.

The plant oncogene rolD stimulates flowering in transgenic tobacco plants.

The Agrobacterium rhizogenes T-DNA oncogene rolD under the control of its own 5' regulatory region was transferred to day-neutral tobacco plants. The main trait induced by rolD in transgenic plants is a striking precocity in flower setting and a strong enhancement of the flowering potential. In rolD plants, early flowering is followed by the very rapid growth of numerous lateral inflorescences. The analysis of several morphological and histological parameters suggests that some characteristic morphological abnormalities observed in rolD plants can be accounted for by their early reproductive phase transition and points to the involvement in the transition of a greater portion of the plant body than is the case for untransformed tobacco. The in vitro morphogenic potential of tissues from rolD plants was also tested. Superficial thin cell layer explants from rolD plants show an earlier and much enhanced flower organogenesis, compared to controls, both on flowering and on hormone-free medium.

Oligogalacturonides Prevent Rhizogenesis in rolB-Transformed Tobacco Explants by Inhibiting Auxin-Induced Expression of the rolB Gene.

Oligogalacturonides elicit several defense responses and regulate different aspects of growth and development in plants. Many of the development-related effects of oligogalacturonides appear to be amenable to an auxin antagonist activity of these oligosaccharins. To clarify the role of oligogalacturonides in antagonizing auxin, we analyzed their effect on root formation in leaf explants of tobacco harboring the plant oncogene rolB. We show here that oligogalacturonides are capable of inhibiting root morphogenesis driven by rolB in transgenic leaf explants when this process requires exogenous auxin. Because rolB expression is induced by auxin and dramatically alters the response to this hormone in transformed plant cells, the inhibiting effect of oligogalacturonides could be exerted on the induction of rolB and/or at some other auxin-requiring step(s) in rhizogenesis. We show that oligogalacturonides antagonize auxin primarily because they strongly inhibit auxin-regulated transcriptional activation of a rolB-[beta]-glucuronidase gene fusion in both leaf explants and cultured leaf protoplasts. In contrast, oligogalacturonides do not inhibit rhizogenesis when rolB transcriptional activation is made independent of auxin, as shown by the lack of inhibition of root formation in leaf explants containing rolB driven by a tetracycline-inducible promoter.

Cytological events induced by arachidonic acid in potato tubers.

Necrosis of potato tuber cells is enhanced, after 24 h incubation, by arachidonic acid (AA), an elicitor of the hypersensitive response in Solarium tuberosum L. and also by Fenton's reagents (a hydroxyl radical-generating system) either alone or in association with AA. The phenomenon is independent of tuber age. In aged tubers, phenylthiourea (PTU) causes a significant reduction of AA-induced cell necrosis. Necrosis observed in the presence of PTU alone is lower than in other treatments, and neither different from the controls nor affected by-tuber age. Cell size is not affected by treatments or ageing. Nuclear hypertrophy occurs independently of tuber ape, with the highest values after treatment with AA and Fenton's reagents. Nucleolar extrusion is observed in all treatments but earliest in the presence of AA. AA also enhances the number of lignified parenchymal cells and, to a lesser extent, the number of traeheary elements.