Salt tolerance analysis of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> NOK2 accession under saline conditions and potassium supply

The response to salt treatment and K⁺ provision of two Arabidopsis thaliana accessions grown for 17 days in the presence of 50 mM NaCl was investigated. Leaf and root dry weight deposition was restricted by salt, more in Col accession than in NOK2 accession. In both accessions, the growth inhibition induced by salinity was associated with a decrease in total leaf surface area, which resulted from diminished leaf number, but not from restriction of individual leaf surface area. Comparing the effects of salt on dry matter production and total leaf surface area revealed large difference between Col and NOK2 for net assimilation rate (the amount of whole plant biomass produced per unit leaf surface area), which was augmented by salt and K⁺ in NOK2 but not in Col. This result, which suggested a better capacity of NOK2 to preserve its photosynthetic machinery against salt stress, was in agreement with the effect of NaCl on photosynthetic pigments. Indeed, salt significantly reduced chlorophyll and carotenoid content in Col leaves but had no impact on NOK2 leaf pigment content. Since K⁺ provision had only marginal effects on these responses to salt stress, leaf mineral unbalance was unlikely. Guaiacol peroxidase activity was augmented by salt treatment in leaves and roots of both accessions. Salinity decreased the catalase activity in Col leaves and in roots, and increased this activity in NOK2 organs. In conclusion, when aggressed by salt, NOK2 was able (1) to produce more leaves than Col, and (2) to efficiently protect its photosynthetic apparatus, perhaps by developing more efficient antioxidative defense through increased catalase and peroxidase activities. Consequently, the overall photosynthetic activity was higher and more robust to salt aggression in NOK2 than in Col.     

A strategy of Ca2+ alleviating Na+ toxicity in salt-treated Cyclocarya paliurus seedlings: photosynthetic and nutritional responses

Cyclocarya paliurus seedlings were subjected to 85?mM NaCl and 0, 6, 12 or 18?mM Ca(NO₃)₂ treatments to study changes in plant growth, photosynthetic parameters and distribution and/or accumulation of organic and inorganic solutes. Na⁺ toxicity symptoms were observed in plants non-treated with Ca(NO₃)₂, while 12?mM Ca(NO₃)₂ supplementation produced a significant promotion of shoot growth; meanwhile chlorophyll content, photosynthetic rate and optimum quantum yield of photosystem II (PSII), represented by the F_v/F_m ratio and pigments content as well as proline and soluble sugars, significantly increased. Ca(NO₃)₂ supply increased K⁺ and Ca²⁺ concentration, whereas the Na⁺ transport to the shoot was inhibited. There was a strong increase in the K⁺/Na⁺ ratio in shoot of Ca(NO₃)₂-treated plants. X-Ray microanalysis of roots showed that K⁺, Ca²⁺ and Na⁺ accumulated mainly in the epidermal cells and cortical cells of roots with 12?mM Ca(NO₃)₂ supply, and low accumulation was observed in stelar parenchyma, indicating exogenous Ca²⁺ possibly induced or strengthened effects of Casparian bands on ion transport. These results suggest that Ca(NO₃)₂ supplement increased inorganic and organic solutes accumulation in shoot and leaf, and restricted Na⁺ transport to the shoot by reinforcing barrier effects for attenuating salt injuries in plants, which could be a strategy of Ca²⁺ alleviating Na⁺ toxicity in C. paliurus seedlings subjected to salt stress.     

Increased cucumber salt tolerance by grafting on pumpkin rootstock and after application of calcium

Self-grafted and pumpkin rootstock-grafted cucumber plants were subjected to the following four treatments: 1) aerated nutrient solution alone (control), 2) nutrient solution with 10 mM Ca(NO₃)₂ (Ca), 3) nutrient solution with 90 mM NaCl (NaCl), and 4) nutrient solution with 90 mM NaCl + 10 mM Ca(NO₃)₂ (NaCl+Ca). The NaCl treatment decreased the plant dry mass and content of Ca²⁺ and K⁺ but increased the Na⁺ content in roots and shoots. Smaller changes were observed in pumpkin rootstock-grafted plants. Supplementary Ca(NO₃)₂ ameliorated the negative effects of NaCl on plant dry mass, relative growth rate (RGR), as well as Ca²⁺, K⁺, and Na⁺ content especially for pumpkin rootstock-grafted plants. Supplementary Ca(NO₃)₂ distinctly stimulated the plasma membrane (PM) H⁺-ATPase activity which supplies the energy to remove excess Na⁺ from the cells. The expressions of gene encoding PM H⁺-ATPases (PMA) and gene encoding a PM Na⁺/H⁺ antiporter (SOS1) were up-regulated when Ca(NO₃)₂ was applied. The pumpkin rootstock-grafted plants had higher PM H⁺-ATPase activity as well as higher PMA and SOS1 expressions than the self-grafted plants under NaCl + Ca treatment. Therefore, the addition of Ca²⁺ in combination with pumpkin rootstock grafting is a powerful way to increase cucumber salt tolerance.     

The effects of calcium sulphate on growth,membrane stability and nutrient uptake of tomato plants grown under salt stress

A pot experiment was carried out with tomato (Lycopersicon esculentum Mill.) cv. “Target F1” in a mixture of peat, perlite, and sand (1:1:1) to investigate the effects of supplementary calcium sulphate on plants grown at high NaCl concentration (75 mM). The treatments were: (i) control (C), nutrient solution alone; (ii) salt treatment (C + S), 75 mM NaCl; (iii) salt plus calcium treatment 1 (C + S + Ca1), 75 mM NaCl plus additional mixture of 2.5 mM CaSO₄ in nutrient solution; (iv) salt plus calcium treatment 2 (C + S + Ca2), 75 mM NaCl plus additional mixture of 5 mM CaSO₄ in nutrient solution. The plants grown under salt stress produced low dry matter, fruit weight, and relative water content than those grown in standard nutrient solution. Supplemental calcium sulphate added to nutrient solution containing salt significantly improved growth and physiological variables affected by salt stress (e.g. plant growth, fruit yield, and membrane permeability) and also increased leaf K⁺, Ca²⁺, and N in tomato plants. The effects of supplemental CaSO₄ in maintaining membrane permeability, increasing concentrations of Ca²⁺, N, and K⁺ and reducing concentration of Na⁺ (because of cation competition in root zone) in leaves could offer an economical and simple solution to tomato crop production problems caused by high salinity.     

Physiological and molecular characterization of salt response of <Emphasis Type="Italic">Arabidopsis thaliana NOK2</Emphasis> ecotype

Arabidopsis thaliana is a glycophyte capable to tolerate mild salinity. Although salt sensitivity of this species, a variability of this characteristic was revealed between different ecotypes. This study presents the physiological and molecular characteristics of salt response of two ecotypes, NOK2 and Columbia (Col). Seedlings were cultivated in hydroponics in the presence of 0 or 50 mM NaCl during 25 days. Rosette leaf samples were collected after 19, 22, and 25 days for determination of physiological parameters, and after 18 days for study of DNA polymorphism. Salt treatment decreased rosette dry matter, leaf number, leaf hydration, and leaf surface area. All these effects were significantly more visible in Col than in NOK2. Moreover, the NOK2 leaves accumulated less Na⁺ and more K⁺ than those of Col. DNA polymorphism between the two ecotypes was analyzed with codominant molecular markers based on PCR amplification, namely, microsatellites, cleaved amplified polymorphism sequence (CAPS), and single nucleotide polymorphism markers (SNP). Among the 35 tested markers, 17 showed a clear polymorphism and were distributed on the five Arabidopsis chromosomes ending with a genetic map construction. These results could play an important role in the future establishment of cartography of candidate gene controlling the K⁺/Na⁺ selectivity of ion transport in leaves, a component of plant salt tolerance.     

H2O2 and cytosolic Ca2+ signals triggered by the PM H+‐coupled transport system mediate K+/Na+ homeostasis in NaCl‐stressed Populus euphratica cells

Using confocal microscopy, X‐ray microanalysis and the scanning ion‐selective electrode technique, we investigated the signalling of H₂O₂, cytosolic Ca²⁺ ([Ca²⁺]_cyt) and the PM H⁺‐coupled transport system in K⁺/Na⁺ homeostasis control in NaCl‐stressed calluses of Populus euphratica. An obvious Na⁺/H⁺ antiport was seen in salinized cells; however, NaCl stress caused a net K⁺ efflux, because of the salt‐induced membrane depolarization. H₂O₂ levels, regulated upwards by salinity, contributed to ionic homeostasis, because H₂O₂ restrictions by DPI or DMTU caused enhanced K⁺ efflux and decreased Na⁺/H⁺ antiport activity. NaCl induced a net Ca²⁺ influx and a subsequent rise of [Ca²⁺]_cyt, which is involved in H₂O₂‐mediated K⁺/Na⁺ homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na⁺/H⁺ antiport system, the NaCl‐induced elevation of H₂O₂ and [Ca²⁺]_cyt was correspondingly restricted, leading to a greater K⁺ efflux and a more pronounced reduction in Na⁺/H⁺ antiport activity. Results suggest that the PM H⁺‐coupled transport system mediates H⁺ translocation and triggers the stress signalling of H₂O₂ and Ca²⁺, which results in a K⁺/Na⁺ homeostasis via mediations of K⁺ channels and the Na⁺/H⁺ antiport system in the PM of NaCl‐stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.     

Salt-imposed restrictions on the uptake of macroelements by roots of Arabidopsis thaliana

The aim of this investigation was to identify the growth limiting factors in Arabidopsis thaliana subjected to a mild salt stress. Two natural accessions (Col and N1438) were compared. In spite of their morphological and developmental similarity, they have been previously shown to differ in the response of their superoxide dismutase genes to salt stress (Physiol Plant 132:293–305, 2008). Thirty-day-old seedlings were grown for 15 days using a split-root configuration in which the root system was divided into two equal parts: the first was immersed in a complete nutrient solution with 50 mM NaCl added, while the second part was immersed in either complete or incomplete K-, Ca-, or N-free medium. Using this approach, we demonstrated that K⁺ and Ca²⁺ uptake was impaired in the roots subjected to NaCl. There was no indication of the salt-induced inhibition of N uptake. If K⁺ or Ca²⁺ were available from salt-free medium, plants were able to grow at normal rate and accumulate large amounts of Na⁺ in the shoots. These results indicate that the sensitivity of Arabidopsis growth to mild salinity was probably due to an inhibition of K⁺ or Ca²⁺ root transport by salt rather than due to salt accumulation in shoots. Furthermore, the salt sensitivity of ion transport in roots seemed to depend on the genotype, since K⁺ was limiting for Col growth, in contrast to N1438, the growth of which was limited by Ca²⁺.     

Different NaCl-Induced Calcium Signatures in the Arabidopsis thaliana Ecotypes Col-0 and C24

A common feature of stress signalling pathways are alterations in the concentration of cytosolic free calcium ([Ca²⁺]_cyt), which allow the specific and rapid transmission of stress signals through a plant after exposure to a stress, such as salinity. Here, we used an aequorin based bioluminescence assay to compare the NaCl-induced changes in [Ca²⁺]_cyt of the Arabidopsis ecotypes Col-0 and C24. We show that C24 lacks the NaCl specific component of the [Ca²⁺]_cyt signature compared to Col-0. This phenotypic variation could be exploited as a screening methodology for the identification of yet unknown components in the early stages of the salt signalling pathway.     

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.     

披针叶黄华试管苗适应盐胁迫的渗透调节机制

以披针叶黄华(Thermopsis lanceolata)试管苗为材料,通过组培方法研究其在0、0.2%、0.4%、0.6%、0.8%和1.0%NaCl和Na2SO4胁迫30d后的生长、有机渗透调节物质和无机渗透调节物质(Na+、K+和Ca2+)含量的变化,以探讨其耐盐性机制。结果显示:(1)随NaCl和Na2SO4胁迫浓度的增加,披针叶黄华试管苗叶片脯氨酸和可溶性糖含量均显著持续增加,且NaCl胁迫下脯氨酸上升的幅度均大于相同浓度Na2SO4胁迫下的增幅,而可溶性糖上升的幅度却小于相同浓度Na2SO4胁迫下的幅度;可溶性蛋白含量随NaCl浓度的增大呈先升高后降低的趋势,但随Na2SO4浓度的增加呈持续上升的趋势。(2)随NaCl和Na2SO4浓度的增加,披针叶黄华试管苗Na+含量呈增加趋势且各处理均显著高于对照,Ca2+含量和叶片K+含量却呈逐渐减少趋势且各处理均显著低于对照,而根系K+含量呈先降后升的趋势;Na2SO4胁迫下披针叶黄华试管苗叶片Na+含量上升幅度以及K+和Ca2+含量下降幅度均明显低于相同浓度NaCl胁迫组;而Na+/K+和Na+/Ca2+比值随NaCl和Na2SO4浓度增加而升高;NaCl胁迫下,叶片Na+/K+和Na+/Ca2+高于相同浓度Na2SO4胁迫下的比值,而根系Na+/K+和Na+/Ca2+却低于相同浓度Na2SO4胁迫下的比值。研究表明,盐胁迫下,披针叶黄华试管苗通过抑制叶片中Na+积累并增加可溶性糖和可溶性蛋白含量,在根系中维持较高K+和Ca2+含量以及较低水平Na+/K+和Na+/Ca2+比,以降低披针叶黄华细胞渗透势来适应盐渍环境;披针叶黄华对NaCl胁迫的调节能力弱于Na2SO4。     

Amelioration of salt-induced oxidative stress in eggplant by application of 24-epibrassinolide

The effects of exogenous 24-epibrassinolide (EBR) on the growth, oxidative damage, antioxidant system and ion contents in eggplant (Solanum melongena L.) seedlings under salt stress were investigated. Eggplant seedlings were exposed to 90 mM NaCl with 0, 0.025, 0.05, 0.10 and 0.20 mg dm⁻³ EBR for 10 d. EBR, especially at concentration 0.05 mg dm⁻³, alleviated growth suppression caused by NaCl stress, decreased electrolyte leakage, superoxide production and content of malondialdehyde and H₂O₂ in NaCl-treated plants. EBR also increased activities of superoxide dismutase, guaiacol peroxidase, catalase and ascorbate peroxidase and the contents of ascorbic acid and reduced glutathione. Furthermore, we also found that Na⁺, Cl⁻ contents were decreased, K⁺, Ca²⁺ contents and K⁺/Na⁺, Ca²⁺/Na⁺ ratios were increased in the presence of EBR under salt stress.     

Contribution of <Emphasis Type="Italic">Glomus intraradices</Emphasis> inoculation to nutrient acquisition and mitigation of ionic imbalance in NaCl-stressed <Emphasis Type="Italic">Trigonella foenum-graecum</Emphasis>

The study aimed to investigate the effects of an AM fungus (Glomus intraradices Schenck and Smith) on mineral acquisition in fenugreek (Trigonella foenum-graecum) plants under different levels of salinity. Mycorrhizal (M) and non-mycorrhizal (NM) fenugreek plants were subjected to four levels of NaCl salinity (0, 50, 100, and 200 mM NaCl). Plant tissues were analyzed for different mineral nutrients. Leaf senescence (chlorophyll concentration and membrane permeability) and lipid peroxidation were also assessed. Under salt stress, M plants showed better growth, lower leaf senescence, and decreased lipid peroxidation as compared to NM plants. Salt stress adversely affected root nodulation and uptake of NPK. This effect was attenuated in mycorrhizal plants. Presence of the AM fungus prevented excess uptake of Na⁺ with increase in NaCl in the soil. It also imparted a regulatory effect on the translocation of Na⁺ ions to shoots thereby maintaining lower Na⁺ shoot:root ratios as compared to NM plants. Mycorrhizal colonization helped the host plant to overcome Na⁺-induced Ca²⁺ and K⁺ deficiencies. M plants maintained favorable K⁺:Na⁺, Ca²⁺:Na⁺, and Ca²⁺:Mg²⁺ ratios in their tissues. Concentrations of Cu, Fe, and Zn²⁺ decreased with increase in intensity of salinity stress. However, at each NaCl level, M plants had higher concentration of Cu, Fe, Mn²⁺, and Zn²⁺ as compared to NM plants. M plants showed reduced electrolyte leakage in leaves as compared to NM plants. The study suggests that AM fungi contribute to alleviation of salt stress by mitigation of NaCl-induced ionic imbalance thus maintaining a favorable nutrient profile and integrity of the plasma membrane.     

Salt Tolerance in Aquatic Macrophytes: Ionic Relation and Interaction

Effects of seawater salinity (SWS) and pure NaCl on the intracellular contents of Na⁺, K⁺, Mg²⁺, Ca²⁺, chlorophylls (Chl) and carotenoids (Car) were studied in three submerged aquatic macrophytes, Hydrilla verticillata, Najas indica and Najas gramenia, which differed in their tolerance to salinity. NaCl resulted in significant increase in Chl/Car ratio in the salt-sensitive H. verticillata and moderately salt-tolerant N. indica, but not in the salt-tolerant N. gramenia. SWS treatment did not result in any significant change in the ratio. The intracellular content of Na⁺ increased significantly in all the test plants upon exposure to both NaCl and SWS. The content of K⁺ decreased significantly in these plants upon salinity treatment, except in N. gramenia. The contents of Ca²⁺ and Mg²⁺ decreased significantly upon NaCl treatment and remained unchanged or increased upon SWS treatment. No relationship between salt tolerance and K⁺/Na⁺ ratio was observed. The maintenance of a minimal level of K⁺ was observed to be the most probable requirement of salt tolerance in aquatic macrophytes.     

NaCl胁迫对圆柏幼苗生长和离子吸收及分配的影响

以当年生圆柏幼苗为实验材料,采用温室调控盆栽土培法研究了不同浓度NaCl(0、100、200、300mmol·L-1)胁迫21d对其生长情况及不同器官(根、茎、叶)中K~+、Na~+、Ca~(2+)和Mg~(2+)的吸收和分配的影响,以探讨圆柏幼苗对盐环境的生长适应性及耐盐机制。结果表明:(1)随着NaCl胁迫浓度的增加,圆柏幼苗生长,包括株高、地径、相对生长量以及生物量的积累均呈下降趋势,而其根冠比却增加。(2)在各浓度NaCl胁迫处理下,圆柏幼苗根、茎、叶中Na~+含量较对照均显著增加,而且叶中Na~+含量显著高于茎和根,叶中Na~+含量是根中的5倍。(3)随着NaCl胁迫浓度的升高,圆柏幼苗各器官中K~+、Ca~(2+)和Mg~(2+)含量以及K~+/Na~+、Ca~(2+)/Na~+及Mg~(2+)/Na~+比值均呈下降趋势。(4)在NaCl胁迫条件下,圆柏幼苗根系离子吸收选择性系数SK,Na、SCa,Na、SMg,Na显著提高,茎、叶离子转运选择性系数SCa,Na、SMg,Na则逐渐降低,叶中离子转运选择性系数SK,Na则随着NaCl胁迫浓度的升高显著降低,大量Na~+进入地上部,减缓了盐胁迫对根系的伤害。研究认为,圆柏幼苗的盐适应机制主要是通过根系的补偿生长效应及茎、叶对Na~+的聚积作用来实现的,同时也与根对K~+、Ca~(2+)、Mg~(2+)的选择性运输能力增强和茎、叶稳定的K~+、Ca~(2+)、Mg~(2+)的选择性运输能力有关。     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

Leaf–water relations and ion concentrations of the halophyte <Emphasis Type="Italic">Atriplex hortensis</Emphasis> in response to salinity and water stress

The impact of salinity and water stress was analyzed in the xero-halophyte Atriplex hortensis using two varieties: green orach (A. hortensis var. purpurea) and red orach (A. hortensis var. rubra). A. hortensis L. is a C₃ species well adapted to salt and drought conditions. To collect information on the physiological impact of different salt and water deficit levels on their water stress resistance, plants were exposed for 3 months to solution containing four levels of NaCl or to water stress regimes including four levels of field capacity. Osmotic potential at zero turgor Ψ_s⁰, osmotic potential at full turgor (Ψ_s¹⁰⁰), relative water content (RWC), ion concentration (Na⁺, K⁺, Ca²⁺, Mg²⁺, and Cl⁻), and malondialdehyde (MDA) were determined at the end of the treatment. The salinity and water stress induced a decrease in Ψ_s¹⁰⁰, Ψ_s⁰, and RWC in both varieties, recorded changes being higher in plants of red variety than those of green variety. Both varieties specifically accumulated Na⁺ in response to drought and salt stress, suggesting that this element could play a physiological role in the stress response of this xero-halophyte species. In contrast, the presence of NaCl and water stress induced a decrease in K⁺, Ca²⁺, and Mg²⁺ concentration in both varieties. Salinity clearly induced an increase in Cl^– concentration in all tissues, but water stress had no impact on this parameter. MDA concentration increased in response to water stress and exogenous NaCl. Based on these findings the more drought-tolerant red orach may be grown in water-limiting soils.     

Growth characteristic and ion contents of rice callus under the influence of NaCl and hydroxyproline

Calli of salt tolerant (Bhoora rata) and salt susceptible (GR₁₁) rice varieties were cultured on Linsmaeir and Skoog’s medium containing LD₅₀ concentration of NaCl (200 mM) and hydroxyproline (10 mM). Growth rate of callus and Na⁺, K⁺, Cl⁻, Mg⁺², and Ca⁺² contents of the cultured rice tissues were determined at the end of 0, 2, 4 and 6 weeks of incubation. Hydroxyproline resistant calli of both rice varieties when cultured on Linsmaeir and Skoog’s medium containing both NaCl and hydroxyproline showed increased dry weight and enhanced intracellular levels of K⁺, Mg⁺² and Ca⁺². The accumulation of Na⁺ and Cl⁻ ions was less in the hydroxyproline resistant calli.