Analysis of secreted protease inhibitors after water stress in potato tubers

Potato tubers (Solanum tuberosum) secrete two kinds of proteinase inhibitors after a water stress. The polypeptides have differing inhibitory activities but are Kunitz-type inhibitors based on amino-terminal sequences homologies. A proteolysis maturation type of a cell protease inhibitor was observed. They can constitute high MW complex, sometimes with another type of protein. The function of these protease inhibitors is discussed in relation to plant defence.     

Effect of water stress on proline catabolism in tobacco leaves

Proline-[¹⁴C] infiltrated into leaf disks of tobacco (Nicotiana tabacum cv BY-4) in the dark was converted to glutamic acid and then metabolized through the TCA cycle. A smaller amount of proline-[¹⁴C] was metabolized when the leaf disks were wilted than when turgid. During a 6 hr period following rehydration, disks converted a larger amount of proline-[¹⁴C] to oxidized products than when wilted, although the proline content of rehydrated disks had not declined. These results indicate that proline oxidation is inhibited by water stress.     

Water deficit enhancement of proline and α-amino nitrogen accumulation in potato plants and its association with susceptibility to drought

Potato plants ( Solanum tuberosum L. cvs 'Up-to-Date', 'Desiree', 'Alpha', 'Spunta', 'Elvira' and 'Troubadour') were exposed to cycles of water stress and relief during growth. Severe water deficit induced increased proline content 6- to 7-fold in nonturgid leaves which just started to wilt, and 8- to 27-fold in fully wilted leaves of potatoes. However, proline content was not affected during the early stages of stress development over a range of osmotic potentials in the leaves. The rising proline content was related to turgor loss of leaves independent of changes in the osmotic potentials, which indicates that proline involvement in osmoregulation of potato leaves is unlikely.
Repeated cycles of water stress and relief resulted in increased proline and α-amino nitrogen content in the tuber tissue of some of the cultivars. The smallest increase in proline content was obtained in 'Alpha' tubers and the content of α-amino nitrogen remained unaffected by the water stress. Concomitantly, 'Alpha' was the most drought-tolerant cultivar, as determined by its capacity to accumulate dry matter in tubers under stress conditions. On the other hand, in tubers of cultivars which were more susceptible to drought, a marked increase in proline and α-amino nitrogen was observed in response to water stress. The possible association of these findings with tolerance of potatoes to repeated short periods of drought is discussed.     

水分胁迫对小麦幼苗及悬浮培养细胞中诱导蛋白表达的影响

丰抗8号小麦幼苗及成熟胚诱导的悬浮培养细胞在水分胁迫（－1．0MPa PEG6000）下,可溶性蛋白含量与蛋白组分变化有差异,幼苗可溶性蛋白含量高于对照,并随生长的延长呈降低趋势;悬浮培养细胞可溶性蛋白含量低于对照,且略有上升;复水后均可恢复对照水平,SDS－PAGE电泳及薄层扫描分析结果表明,幼苗受水分胁迫诱导,出现44．2kD蛋白亚基,该蛋白亚基含量可随胁迫时间延长上升,复水后消失,在正常条件下悬浮培养细胞中含有44．2kD蛋白亚基表达,轻度胁迫处理时,该蛋白亚基含量上升,对悬浮培养细胞进行水分胁迫,该蛋白则表现下降趋势,复水后又可上升。     

Thermal stress evaluation of suspension cell cultures in winter wheat

Summary The objectives of this study were to compare thermotolerance in whole plants vs. suspension cell cultures of winter wheat, and to evaluate the synthesis of heat shock proteins in relation to genotypic differences in thermotolerance in suspension cells. Whole plant genetic differences in the development of heat tolerance were identified for three wheat genotypes (ND 7532, KS 75210 and TAM 101). Suspension cell cultures of these genotypes were used to evaluatein vitro response to heat stress. Viability tests by triphenyl tetrazolium chloride (TTC) and by fluorescein diacetate (FD) were utilized to determine the relationship of cellular response to heat stress (37°C/24 h, 50°C/1h). KS 75210 and ND 7532 are relatively heat susceptible. TAM 101 is heat tolerant. Both tests at the cellular level were similar to the whole plant response. Thus, cellular selection for enhancing heat tolerance seems feasible. Heat shock protein (HSP) synthesis of two genotypes, ND 7532 and TAM 101 were determined for suspension cultured cells. In suspension cultures, HSPs of molecular weight 16 and 17 kD were found to be synthesized at higher levels in the heat tolerant genotype (TAM 101) than the susceptible genotype (ND 7532), both at 34° and 37°C treatments for 2 hours and 5 hours. HSP 22 kD was synthesized more at 34°C for TAM 101 than ND 7532, but not at 37°C; whereas, HSP 33 kD was synthesized at 37°C at similar abundance for both genotypes, but not at 34°C.These results indicated that there is a differential expression of HSP genes in wheat suspension cells at different temperature stress durations and between heat tolerant and heat susceptible genotypes. It appears that the levels of synthesis of HSPs 16 and 17 kD are correlated with genotypic differences in thermal tolerance at the cellular level in two genotypes of wheat.     

Effects of long-term salt stress on antioxidant defence systems, leaf water relations and chloroplast ultrastructure of potato plants

The effect of long-term (30 days) NaCl treatments (100 mM and 200 mM) on the activity of some antioxidant enzymes, level of antioxidant metabolites, water relations and chloroplast ultrastructure, was studied in potato (Solanum tuberosum cv. Désirée) leaves. Salt stress negatively affected relative water content, leaf stomatal conductance and transpiration rate. In treated plants, proline was enhanced, but there was a significant decrease in ascorbate and proteins. Total superoxide dismutase activity was increased. The isozyme patterns detected in native gels from salt-irrigated plants were not changed although all the isoforms appeared more heavily stained due to higher activity. In contrast, at both levels of NaCl, catalase activity decreased and ascorbate peroxidase activity showed no significant change in comparison with an untreated control. At ultrastructural level, only thylakoid swelling and a decrease in the amount of grana stacking was observed in treated plants. The overall behaviour of the antioxidant enzymes suggests an increase of cellular H₂O₂ that would contribute to the oxidative stress of potato plants, but which may be alleviated somewhat by the enhanced levels of proline.     

Effect of exogenous steroid glycosides on cultured cells of potato under oxidative stress

Low doses of furostanol glycosides (FG) were shown to elevate the activity of peroxidases (guaiacol-dependent and ascorbate peroxidases) and reduce peroxidation of lipids (POL) below the control level in the cell culture of potato (Solanum tuberosum L.). Under oxidative stress (OS) induced by paraquat, FG protected the cell culture from injury with peroxidase activity being high and POL level lower as compared with the effect of paraquat alone. FG did not affect the activity of superoxide dismutase and catalase. Dynamics of the levels of chlorophyll (a + b) and carotenoids depended not only on the effect of FG and paraquat but on the composition of cell population as well. Greenish tissue contained more pigments and was more resistant to the herbicide action than whitish tissue was. Possible reasons for the elevation of resistance of the cultured cells treated with FG under OS are discussed as well as similarity and differences in the responses of cells to the effect of inducers.     

The effect of calcium on flavanol production in cell suspension cultures of Polygonum hydropiper

Cultured Polygonum hydropiper cells maintained in Murashige and Skoog (MS) medium supplemented with 10^–6 m 2,4-D, 10^–6 m kinetin, 0.1% casamino acids and 3% sucrose were transferred to medium containing a higher concentration of calcium chloride (15 mm). The content of flavanols in the cells on the 6th day was approximately twice that of the control culture (31.9–60.7 mg/g dry wt). However, the contents of other secondary metabolites such as chlorogenic acid and gallic acid were not changed. The levels of flavanols in the culture medium remained unchanged throughout the 21-day culture period. Of the the inorganic components supplemented to the culture medium , only elevated levels of calcium chloride induced an increase in flavanol contents of the cells. The results indicated that the elevated concentration of calcium in the culture medium played an important role in activating the accumulation of flavanols. Received: 4 June 1998 / Revision received: 30 October 1998 / Accepted: 29 November 1998     

Frost tolerance in cell cultures of potato

Different methods of freezing and of estimating frost damage in cell cultures of Solanum tuberosum L. and a number of wild Solanum species were compared. Frost-killing temperatures (FKT, i.e. the temperature resulting in 50% of the maximum possible frost damage) in leaves of these species were -6°C ( S. acaule ), 5°C ( S. me-gistacrotobum ), -4.5°C ( S. commersonii ) and -3°C ( S. polytrichon and S. tubero-sum ) No appreciable species differences were found in FKT when cells were submerged in either buffer or medium and frozen. However, differences did exist when cells were frozen in a non-submerged condition: S. acaule and S. commersonii callus were more sensitive to frost than suspensions, whereas suspensions of the other species were the most sensitive. Measurement of freezing damage by either electrolyte leakage or by 2,3,5-triphenyltetrazolium chloride viability assays revealed similar FKT values. Cell cultures of S. acaule showed better frost tolerance than S. tuberosum (FKT values were -4.5 to -6°C and -2.5°C. respectively), however, frost tolerance of S. megistacrolobum and S. commersonii was only poorly expressed at the cell level (FKT values were between -2 and -3°C). Variant cell lines previously selected for resistance to the amino acid analogues hydroxyprotine, aminoethylcysteine and 5-methyhryptophan appeared to be more tolerant to frost than the wild type S. tuberosum clone.     

A review of the physiology of potato tuber dormancy

A review of the scientific literature relating to the physiology of potato (Solanum tuberosum) tuber dormancy is presented. Effort has been concentrated on an up-to-date overview of the current state of understanding, rather than comprehensively covering the very extensive literature going back over many decades. The format chosen follows the fate of the crop. After defining tuberisation and dormancy, the physiological activity of the dormant tuber is reviewed and the storage environment is considered from both a physical and chemical standpoint. Advances in chemical control and the potential for molecular biology are highlighted.     

The effect of NaCl on antioxidant enzyme activities in potato seedlings

The effect of NaCl on the growth and activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were investigated in the seedlings of four potato cultivars (Agria, Kennebec; relatively salt tolerant, Diamant and Ajax; relatively salt sensitive). The shoot fresh mass of Agria and Kennebec did not changed at 50 mM NaCl, whereas in Diamant and Ajax it decreased to 50 % of that in the controls. In Agria and Kennebec, SOD activity increased at 50 mM NaCl, but no significant changes observed in Diamant and Ajax. At higher NaCl concentration, SOD activity reduced in all cultivars. CAT and POD activities increased in all cultivars under salt stress. Unlike the other cultivars, in Ajax seedlings, APX activity increased in response to NaCl stress. We also observed new POD and SOD isoenzyme activities and changes in isoenzyme compositions under salt stress. These results suggest that salt-tolerant potato cultivars may have a better protection against reactive oxygen species (ROS) by increasing the activity of antioxidant enzymes (especially SOD) under salt stress.     

Age of potato seed-tubers influences protein synthesis during sprouting

The effect of seed-tuber age on the ability of tuber tissue to synthesize protein during sprouting was examined. As seed-tuber age advanced from 4 to 32 months (at 4°C, 95% relative humidity), soluble protein concentration of tubers decreased linearly, with a concomitant increase in free amino acid concentration. The age-induced loss of tuber protein may thus be due to increased proteolysis, decreased protein synthesis, or both. Five- and 17-month-old seed-tubers were compared for their ability to incorporate radiolabeled amino acids into soluble protein at equivalent stages of sprout development. Tuber respiration was profiled through each sprouting stage to characterize the physiological status of the seed-tubers prior to incorporation studies. Five-month-old seed-tubers maintained a constant rate of respiration during sprouting. In contrast, respiration of 17-month-old tubers increased as sprout dry matter increased, resulting in a 2- to 3-fold greater respiratory rate from the older tubers, relative to the younger tubers, at similar stages of sprout development. Prior to sprouting, the rate of incorporation of amino acids into trichloroacetic acid-precipitable protein of tissue from 5-month-old tubers was 2. 9-fold higher than that from 17-month-old tubers. More importantly, protein-synthetic capacity of tissue from younger tubers increased about 1. 7-fold during sprout development. Despite the higher respiratory activity and faster total sprout dry matter accumulation from older seed-tubers, protein synthesis remained at a low and constant level through all stages of sprouting. Protein-synthetic capacity thus declines with advancing tuber age, and this may contribute to reduced growth potential during the latter stages of establishment by affecting the ability of seed-tubers to synthesize enzymes involved in mobilization and translocation of tuber reserves to developing plants.     

Influence of osmotic stress on the respiration of potato tuber callus

Potato tuber ( Solanum tuberosum L. cv. Bintje) callus shows a decrease in fresh weight and an increase in dry weight upon transfer to nutrient medium supplemented with 0.3 or 0.5 M mannitol. The osmolarity of the intracellular fluid increases simultaneously. Probably mannitol is taken up from the medium till the osmolarity of the tissue is in equilibrium with that of the medium. After osmotic adaptation, on a medium with 0.5 M mannitol, growth is negligible, although the tissue retains its viability.
Respiration increases upon transfer to medium with extra mannitol, especially when expressed on a fresh weight basis. On this basis cytochrome and alternative pathway capacities do not change appreciably. The respiratory increase is exclusively caused by an increased engagement of the alternative pathway. The participation of this pathway in uninhibited respiration increases from about 10 to 90% upon transfer to medium with extra mannitol. The increase in respiration is partly correlated with the decrease in fresh weight upon transfer. Per disc, the capacities of the cytochrome and alternative pathway decline. Yet, total respiration per disc significantly increases due to the increased participation of the alternative pathway. This results in an almost equal ATP-production per disc before and after transfer. We suggest, that the alternative pathway functions as a reserve capacity in potato callus, which is switched on when ATP-production coupled to the cytochrome pathway is impaired.     

The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.     

Influence of proline and hydroxyproline on salt-stressed axillary bud cultures of two varieties of potato (Solanum tuberosum)

Summary The effects of exogenously supplied proline and hydroxyproline on the potato (Solanum tuberosum) varieties L.T.8 and Desiree were studied using axillary bud cultures both in the presence and absence of 0.6% salt stress. In both varieties, the effects of exogenously supplied proline and hydroxyproline at 1.0 mM and 2.5 mM were less severe than 0.6% salt alone. At the same time, the accumulation of proline, protein, carbohydrates, sodium, and potassium were similar. However, when both the salt and proline/hydroxyproline were supplied, proline and hydroxyproline provided some measure of protection against salt stress. It is believed that increased proline levels in L.T.8 and increased carbohydrates in Desiree due to the presence of exogenously supplied proline/hydroxyproline were responsible for the additional protection against salt stress in the axillary bud cultures of these varieties.     

Molecular mapping of the potato virus Y resistance gene Rysto in potato

Ry _sto is a dominant gene which confers resistance to potato virus Y (PVY) in potato. We have used bulked segregant analysis of an F₁ tetraploid potato population to identify three AFLP markers linked to and on either side of Ry _sto . The tomato homologue of one of these AFLP markers was assigned to linkage group XI by analysis of an F₂ mapping population of tomato, suggesting that Ry _sto is also on chromosome XI of the potato genome. This map position was confirmed by the demonstration that Ry _sto was linked to markers which had been previously mapped to chromosome XI of the potato genome. Four additional AFLP markers were identified that were closely linked to Ry _sto in a population of 360 segregating progeny of a potato cross between a resistant (Ry _sto ) and a susceptible parent. Two of these markers were on either side of Ry _sto , separated by only a single recombination event. The other two markers co-segregated with Ry _sto . Received: 29 July 1996 / Accepted: 30 August 1996     

Genome selection within cell cultures of potato and tobacco

The ploidy level of callus cultures, suspensions and cell cultures derived from single cells and protoplasts of Solanum tuberosum L. and Nicotania tabascum L. was analysed with the aim of studying selection processes. Genome selection was tested using chromosome number. Subculture of callus lines mostly resulted in an increase of cytogenetic destabilization, whereas subculture of suspensions led to an increase in nuclei which had the ploidy level of stock plants after an initial period of destabilization. During the course of subculture, the development of a dominant ploidy level was observed in some genotypes. In other genotypes, two dominant ploidy levels were found. Furthermore, the narrowing of the confidence interval indicated stabilizing selection. In genotypes showing a high rate of instability, no development of a dominant genome was observed. The in vitro procedure is of decisive importance for genome selection within cell cultures. The interaction between genetic destabilization and genome selection is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Temporary chromosome number stabilization in potato cell cultures

Cell cultures of Solanum chacoense (monohaploid) and Solanum tuberosum (tetraploid cultivars and parthenogenetically derived dihaploid clones) were found to be highly mixoploid.Relative stabilization of chromosome number at the ploidy level of the original plant material was achieved in microcalli obtained from single cells or small cell colonies (up to about 5 cells) of stock callus lines. This relative stabilization was maintained over three subcultures, which is sufficient for selection procedures. It has been shown that the stabilization can be maintained during a number of further subcultures. Division centers were repeatedly observed in calli characterized by high mitotic activity. As has been shown for the first time there exist significant differences in the ploidy levels of several division centers within one and the same callus. This is of particular importance to callus subculture.