Phytohormones in the formation of crown gall tumors

Crown gall tumors were initiated in a variety of plant species by infection with Agrobacterium tumefaciens strain B6 and the concomitant changes in the tissue levels of phytohormones, mainly indole-3-acetic acid (IAA) and cytokinins, were analyzed. A comparison was made of these hormones with those produced by virulent and avirulent strains of the bacterium in liquid culture and with those of bacteria-free crown gall callus cultures. Specific radioimmunoassays were employed for hormone determinations. An assay for the quantitation of femto-mol amounts of isopentenyladenosine and related cytokinins was newly developed and is described in detail. The results can be summarized as follows: Virulence in strain B 6 is associated with the ability to release trans-zeatin and increased amounts of IAA into the surrounding environment. In many, but not all plants analyzed, the development of crown gall tumors is also associated with a sharp rise in the levels of trans-zeatin-type zytokinins and IAA (e.g., Euphorbia lathyris, Catharanthus roseus). Crown gall calli growing on hormone-free media varied greatly in their cytokinin levels. In a culture of Nicotiana tabacum, both trans-zeatin and isopentenyladenine or related cytokinins were not detected. Thus, tumor growth cannot be explained on the basis of elevated levels of IAA and/or cytokinins alone.Abbreviations ABA abscisic acid - GC-MS gas chromatography-mass spectroscopy - HPLC high pressure liquid chromatography - IAA indole-3-acetic acid - RIA radioimmunoassay - TLC thin layer chromatography Part 19 in the series Use of immunoassay in plant science     

Gibberellic acid regulation of adventitious shoot formation from tuber discs of potato

Summary The formation of adventitious shoots from potato tuber discs explanted onto a modified Murashige and Skoog (MS) medium containingN ⁶-benzylaminopurine (BAP) (3.0 mg/l), and α-naphthaleneacetic, acid (NAA) (0.01 mg/l), was affected by gibberellic acid (GA). The presence of GA in the explant medium was required for shoot formation and 3×10⁻¹⁰ M GA appeared optimum. However, microscopic examination of the tissue protuberances on the surface of the tuber discs from which shoots arose revealed that GA inhibited the formation of shoot meristems. Tuber discs cultured for 6 wk on MS medium containing BAP and NAA without GA did not initiate adventitious shoots that could be determined visually, but microscopic examination of the tissue protuberances revealed the presence of numerous shoot meristems. Subsequent transfer of these tuber discs to medium with GA but without BAP or NAA resulted in the formation of shoots from 100% of the recultrued dises. Thus it appears that although GA inhibits shoot meristem initiation from potato tuber discs, it is required for shoot development once meristems are initiated. This is Journal Paper 8297 of the Purdue University Agricultural Experiment Station. The research was supported by Purdue University Agricultural Experiment Station Program Improvement Funds. Potato tubers were supplied by Wm. Gehring Farms, Inc., Rensselaer, Indiana.     

The effects of abscisic acid on growth and respiratory characteristics of potato tuber tissue discs

Incubation of potato tuber tissue discs on B5 medium supplemented with 1-naphtyl-acetic acid (NAA) led to callus formation, irrespective of the presence of kinetin; without NAA no callus formation occurred. Incubation in the presence of abscisic acid (ABA) reduced the increases in fresh weight and dry weight both in callus-forming and in non-callus-forming tissue. Mitochondrial respiration was lowered by ABA as well. The induction of the alternative, CN-resistant pathway was inhibited by the presence of ABA, especially in mitochondria from non-callus-forming tissue.The in vivo respiration of the callus-forming tissue was higher than that of the non-callus-forming tissue. Total respiration, cytochrome pathway activity and the capacity of the alternative pathway were all lowered in callus-forming tissue by treatment with ABA. The in vivo activity of the alternative pathway was low in all tissue types, especially after ABA-treatment. The slight stimulation by hydroxamates of the oxygen uptake of callus-forming tissue incubated on medium with NAA and ABA indicates the involvement of a hydroxamate-activated peroxidase in the oxygen uptake of this tissue; this peroxidase seemed not to participate in the oxygen uptake of the other tissues types.In non-callus-forming tissue the oxygen uptake of ABA-treated tissue was very low and almost completely resistant to the combined addition of inhibitors of both the cytochrome and the alternative pathway, indicating that the in vivo activity of the mitochondria in the oxygen uptake of the tissue was very low. The possible causes for this ABA-effect are discussed. In non-callus-forming tissue the treatment with ABA creates a situation which is comparable with that observed in intact potato tubers. This situation is characterized by a tissue respiration lower than that of the isolated mitochondria and an alternative pathway capacity that is low or absent.     

Ultrasound-mediated stable transformation of potato tuber discs

Summary Plasmid DNA (pBARGUS), containing the selectable bar gene for resistance to the herbicide Basta, was delivered into potato tuber discs via ultrasonication. Transformed plants were identified by their ability to grow on a medium containing 1mg phosphinothricin/l. Southern hybridization and plant resistance to the application of Basta indicated that a functional bar gene had integrated into potato chromosomal DNA.     

Influence of growth temperature on respiratory characteristics of mitochondria from callus-forming potato tuber discs

The uninhibited respiration of mitochondria, isolated from potato tuber discs (Solanum tuberosum L. cv. Bintje) incubated on a callus-inducing medium at 28°C, is higher than that of mitochondria from tissue incubated at 8°C. This respiration is composed of a CN-sensitive and a CN-resistant part. The capacity of the CN-resistant alternative oxidase pathway is larger in mitochondria from 28°C tissue than in mitochondria from 8°C tissue (35% and 8% of uninhibited respiration, respectively). The alternative pathway is operative both in mitochondria from 28°C tissue and 8°C tissue.

The observed difference in uninhibited respiration, is not only caused by lower values of respiration via the alternative pathway in mitochondria from 8°C tissue, but also by lower values of respiration via the cytochrome pathway.

A positive correlation has been demonstrated between the incubation temperature (ranging from 4-37°C) and the relative capacity of respiration via alternative pathway in the mitochondria. Induction of alternative pathway is not directly correlated with growth (in terms of increase in fresh weight) of the potato tuber discs.

     

植物冠瘿生长调控及相关基因研究进展

阐述了植物冠瘿产生的影响因素及冠瘿的生长和调控,并对冠瘿发生有关的基因进行了综述。     

Effect of growth regulators on proton transport through cytoplasmic membrane of potato tuber cells

Effects of the growth regulators epibrassinolide-694 (EB), gibberellic acid (GA), and abscisic acid (ABA) on the ATP-dependent translocation of H+ through the membranes of plasma membrane vesicles of potato (Solanum tuberosum L.) tuber cells were studied. The ATP-dependent accumulation of H+ in the plasma membrane vesicles from dormant tubers was inhibited by EB and ABA and stimulated by GA. After the break of dormancy, the stimulatory effect of GA increased, the inhibitory effect of ABA decreased, and EB stimulated the accumulation of H+ in the vesicles. The data suggest that the plasma membrane H+ ATPase is a target of phytohormones that regulate the dormancy of potato tubers.     

Effect of ethylene on DNA synthesis in potato tuber discs

The effect of ethylene on DNA synthesis in potato tuber discsinduced by cutting was examined. Continuous presence of ethylenein the ambient atmosphere of the slices lowered the rate ofinduced DNA synthesis by about 50%, but did not alter the timecourse pattern of development of DNA synthesis. RNA and proteinsyntheses were not affected. The inhibitory effect on DNA synthesiswas observed at as low as 0.01 µl/liter and was due tothe specific action of ethylene, not to a non-specific actionof gaseous hydrocarbons. Ethylene also decreased the numberof cells which could synthesize DNA. The results of ethylenetreatment of various durations at various times after cuttingindicate that a process prerequisite for DNA synthesis and susceptibleto ethylene action starts at about 6 hr after cutting and continuesfor only a limited period. (Received July 5, 1976; )     

Endogenous ethylene formation and the development of the alternative pathway in potato tuber discs

Callus-forming discs from potato ( Solanum tuberosum L. cv. Bintje) tubers grown on a nutrient medium containing an auxin and a cytokinin show both a higher ethylene formation and a higher capacity of the mitochondrial alternative pathway than nongrowing discs (on the same medium without auxin and cytokinin). Addition of 1-ami-nocyclopropane-1-carboxylic acid (ACC) to the nutrient medium of non-growing discs results in an enhancement of the ethylene formation as well as the alternative pathway capacity. In callus-forming tissue, the levels of both these parameters can be suppressed by adding aminoethoxyvinylglycine (AVG) to the nutrient medium without affecting growth. The effects on state 3-respiration of ACC (increase) and AVG (decrease) are relatively small. These results suggest that the alternative pathway capacity is controlled to a considerable extent by the endogenous ethylene formation of the tissues.     

Superoxide anion regulates plant growth and tuber development of potato

A higher concentration of H₂O₂ was detected in the sense transgenic potato plant (SS4) with the lily chCu,ZnSOD sequence, whereas higher levels of O₂⁻ was detected in the antisense transgenic plant (SA1) than the WT plant. The elongation growth in SA1 was significantly inhibited by treatment with diphenyleneiodonium, an inhibitor of O₂⁻ generation, and promoted in the SS4 on treatment with herbicide methyl viologen, a generator of apoplastic O₂⁻. Higher concentrations of GAs were detected during plant growth and the early stage of tuberization in SA1. Complete recovery of the above elongation growth and microtuberization pattern in transgenic plants following treatment of GA₃ or an inhibitor of gibberellin synthesis, paclobutrazol, indicate that these changes were mainly caused by active GA levels. In conclusion, a specific ROS (O₂⁻ ) acts as a signal transducer via GA biosynthetic pathways for the regulation of plant growth and tuber development of potato.     

Inhibitory activity of plant growth regulators on Phytophthora palmivora causing cassava tuber rot

Auxins and cytokinins are implicated in a wide variety of developmental and physiological processes in plants. Phytophthora palmivora causes tuber rot in cassava growing regions of Tamil Nadu and Kerala, South India. The in vitro effect of cytokinin, benzyl amino purine (BAP) and auxins, naphthalene acetic acid (NAA) and indole acetic acid (IAA) on P. palmivora mycelium growth was investigated. The inhibitory activity varied among the growth regulators and complete inhibition of the pathogen was observed at 50, 2000 and 2500 ppm by the BAP, IAA and NAA, respectively. The effective growth regulator, BAP was also analysed on tubers before and after the invasion of the pathogen to observe its effect in tuber. Further, it was also checked against the bio-control agent Trichoderma harzianum. The study indicates that the use of BAP could be an important approach in controlling tuber rot pathogen, P. palmivora.     

Bioassay and attributes of a growth factor associated with crown gall tumors

An improved bioassay is described for a factor that promotes tumor growth which was first obtained from extracts of pinto bean leaves with crown gall tumors. Sixteen primary pinto bean leaves per sample are inoculated with sufficient Agrobacterium tumefaciens to initiate about 5 to 10 tumors per leaf and treated with tumor growth factor at day 3 after inoculation. The diameters of 30 to 48 round tumors (no more than 3 randomly selected per leaf) are measured per test sample at day 6. Mean tumor diameter increased linearly with the logarithm of the concentration of tumor growth factor applied. The tumor growth factor was separated by column chromatography from an ultraviolet light-absorbing compound previously reported to be associated with fractions having maximal tumor growth factor activity. Partly purified tumor growth factor showed no activity in a cytokinin bioassay or an auxin bioassay, and negligible activity in gibberellin bioassays. Representatives of these three classes of growth factors did not promote tumor growth. Extracts from crown gall tumors on primary pinto bean leaves, primary castor bean leaves, Bryophyllum leaves, carrot root slices, and tobacco stems showed tumor growth factor activity, whereas extracts from healthy control tissues did not. Extracts from actively growing parts of healthy pinto beans, Bryophyllum, and tobacco, however, showed tumor growth factor activity. Tumor growth factor is proposed to be a normal plant growth factor associated with rapidly growing tissues. Its synthesis may be activated in nongrowing tissues by infection with Agrobacterium sp.     

Photocontrol of chlorogenic acid biosynthesis in potato tuber discs

The appearance of phenylalanine ammonia-lyase activity and the accumulation of chlorogenic acid in potato tuber discs are stimulated by illumination with white light, whereas the appearance of cinnamic acid 4-hydroxylase activity is unaffected by illumination. The photosensitive step in chlorogenic acid biosynthesis may be by-passed by treatment of discs with exogenous supplies of cinnamic acid, whereas treatment of discs with phenylalanine does not isolate the photosensitive step. Therefore, the site of photocontrol of chlorogenic acid biosynthesis in potato tuber discs is the reaction catalysed by phenylalanine ammonia-lyase. Cinnamic acid 4-hydroxylase activity in vitro is unaffected by p-coumaric acid, caffeic acid or chlorogenic acid. Phenylalanine ammonia-lyase activity in vitro is sensitive to inhibition by cinnamic acid. The in vitro properties of the two enzymes are also consistent with the hypothesis that phenylalanine ammonia-lyase rather than cinnamic acid 4-hydroxylase is important in the regulation of chlorogenic acid biosynthesis in potato tuber discs.     

Mechanisms of crown gall formation: T-DNA transfer fromAgrobacterium tumefaciens to plant cells

Agrobacterium tumefaciens harbouring the Ti plasmid incites crown gall tumor on dicotyledonous species. Upon infection of these plants, T-DNA in the Ti plasmid is transferred by unknown mechanisms to plant cells to be integrated into nuclear DNA. WhenAgrobacterium is incubated with protoplasts or seedlings of dicotyledonous plants, circulation of T-DNA and expression ofvir (virulence) genes on the Ti plasmid are induced. The circularization event is efficiently induced by mesophyll protoplasts of tobacco which are highly competent for transformation by the T-DNA, and is also induced by diffusible phenolic compounds excreted from the protoplasts. The circularization and formation of crown gall both require the expression of thevirD locus, one of the induciblevir genes. These results suggest that the circularization of T-DNA reflects one of steps of the T-DNA transfer during formation of crown gall. In contrast to dicotyledonous plants, monocotyledonous plants are thought to be unresponsive to infection byAgrobacterium. We showed that monocotyledonous plants do not excrete diffusible inducers for the expression ofvir genes, while they contain a novel type of a signal substance(s). This inducer is not detected in the exudates of seedlings of monocotyledonous plants, but is found in the extracts from the seedlings, and also those from the seeds, bran and germ of wheat and oats. This finding suggests that T-DNA processing, and possibly its transfer, should take place whenAgrobacterium invades seedlings and seeds of monocotyledonous plants. Recipient of the Botanical Society Award for Young Scientists, 1987.     

The role of polyamines in potato tuber formation

Summary The involvement of free and conjugated polyamines in tuber formation was studied in in vitro cultured node explants ofSolanum tuberosum cv. Superior. Tubers developed from the axillary buds in 100% of the explants cultured in MS medium containing high sucrose levels and supplemented with kinetin (Kin) and chlorocholine chloride (CCC). The addition of growth regulators was not essential for tuber formation, although smaller tubers were formed in the medium devoid of Kin and CCC. Tuber formation was inhibited in about 75% of node explants treated with 0.5 mM difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase. The inhibitory effect of DFMO was almost completely reversed by putrescine addition. Addition of difluoromethylarginine (DFMA), the analogous inhibitor of arginine decarboxylase, had no effect on tuber formation. DFMO, but not DFMA, also inhibited the development of axillary buds into shoots in light-grown node explants. Aminooxyphenylpropionic acid (0.1 to 0.25 mM), an inhibitor of phenylalanine ammonia lyase, caused a sharp reduction in cinnamoyl putrescines, but had no effect on tuber formation. Our results suggest that hydroxycinnamic acids are not causal in tuber formation but may serve as polyamine storage pools. Our findings support the hypothesis that polyamines derived via the ornithine decarboxylase-mediated pathway are necessary for tuber formation in vitro, probably at the early phase of morphogenesis involving active cell division.     

Regeneration of tobacco plants from crown gall tumors

Summary Tissue culture methods have been developed for regeneration of normal appearing tobacco plants from bacteria-free crown gall strains incited byAgrobacterium tumefaciens C58, IIBV7, B6, CGIC, A6NC, 27, and AT4. Regenerants fall into two categories depending on the properties of tissues from these plants. The first type of regenerant was obtained from tumors incited byA. tumefaciens C58 and it retained the potential for expression of tumor characteristics such as a nonrequirement for phytohormones (auxin and cytokinin) by explants in vitro and the presence of detectable concentrations of nopaline. Normal appearing plants obtained from C58 tumors had much lower concentrations of nopaline than the corresponding tumor tissue (130 versus 1700 μg per g dry wt) indicating a parallel repression of abnormal growth and nopaline concentrations in regenerants. The second type of regenerant was obtained from tumors incited by the otherA. tumefaciens strains and was characterized by requirements for phytohormones by explants in vitro and the apparent lack of octopine or nopaline in regenerant tissues.     

Changes in cyclic-AMP phosphodiesterase activity during the formation of crown-gall tumors on potato discs

Cyclic-AMP phosphodiesterase activity was found in crown-galltumor tissue obtained from the inoculation of potato discs withAgrobacterium tumefacicns strain B6. Consistently more enzymeactivity was found, in this tumor tissue than in the correspondingnormal tissue. This difference in enzyme activity was observedwhen strains other than B6 were used to induce tumors, or whennormal and tumor potato tissue grown in culture were examinedfor phosphodiesterase activity. (Received January 27, 1978; )     

Ribosomal RNA synthesis in newly sliced discs of potato tuber

A burst of ribosomal RNA synthesis is induced in potato tissue by slicing, and continues at a decreasing rate for about 12 hours. Ribosomal RNA synthesis in potato discs is sensitive to puromycin, in contrast to non-ribosomal RNA synthesis. Thus, the influence of puromycin on total RNA synthesis is significant only during the first 12 hours following slicing. The function of RNA made after 12 hours in a puromycin-insensitive manner is unknown. However, it is apparently unrelated to protein synthesis, since it has been shown that total inhibition of RNA synthesis by addition of actinomycin D to potato tissue after 12 hours of aging has no effect upon protein synthesis during the ensuing 12 hours.     

Aeromonas, a possible etiological agent in the formation of secondary tumors of crown gall

Abstract From a secondary tumor in a bean stem we have isolated a Gram-negative bacteria, named by us T.2. These bean stems had crown gall tumors induced by the ATV strain of Agrobacterium tumefaciens . This bacterium was classified as belonging to the genus Aeromonas and possesses the capacity of inducing overgrowths in plants, synthesizing indole acetic acid (IAA). The codified phenotypic characteristics of bacterium T.2. via the Ti-plasmid of A. tumefaciens , such as opine utilization and sensitivity to agrocin 84, have been studied. Neither octopine nor nopaline is utilized by T.2. and it is resistant to agrocin 84, whereas the strain ATV of A. tumefaciens utilizes nopaline, and is sensitive to agrocin 84.