盐胁迫下两个甜瓜品种转录因子的转录组分析

利用新一代高通量测序手段——转录组测序（RNA-Seq）技术研究在300 mmol.L-1NaCl胁迫下两个甜瓜（Cucumis melo L.）品种‘玉露’和‘冰雪脆’的转录因子基因表达变化。这两个甜瓜品种在叶绿素荧光参数上的差异表明其在盐胁迫下有不同的生理反应。转录组测序结果表明,盐胁迫与对照相比,‘玉露’共有属于19个转录因子家族的56个转录因子基因表达发生变化（在转录水平,属于7个转录因子家族的22个转录因子上调表达,属于14个转录因子家族的34个转录因子下调表达）。‘冰雪脆’有属于20个转录因子家族的47个转录因子基因表达发生变化（在转录水平上,属于5个转录因子家族的17个转录因子上调表达,属于17个转录因子家族的30个转录因子下调表达）。盐胁迫下,两个甜瓜品种差异表达的转录因子既表现特异性,也存在部分重叠。‘玉露’有29个转录因子特异响应,‘冰雪脆’有20个特异响应。盐胁迫响应重叠的转录因子有27个,其中9个上调表达,18个下调表达。采用实时荧光定量PCR对几个转录因子进行了盐胁迫下的表达检测,其趋势与转录组分析结果基本一致。     

Effects of salinity on uptake and distribution of Na+, Cl- and K+ in two wheat cultivars

Plants of two wheat (Triticum aestivum L.) cultivars differing in salt tolerance were grown in sand with nutrient solutions. 35-d-old plants were subjected to 5 levels of salinity created by adding NaCl, CaCl₂ and Na₂SO₄. Growth reduction caused by salinity was accompanied by increased Na⁺ and Cl^- concentrations, Na⁺/K⁺ ratio, and decreased concentration of K⁺. The salt tolerant cv. Kharchia 65 showed better ionic regulation. Salinity up to 15.7 dS m^-1 induced increased uptake of Na⁺ and Cl^- but higher levels of salinity were not accompanied by further increase in uptake of these ions. Observed increases in Na⁺ and Cl^- concentrations at higher salinities seemed to be the consequence of reduction in growth. Uptake of K⁺ was decreased; more in salt sensitive cultivar. This was also accompanied by differences in its distribution.     

Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na loading and stomatal density

Quinoa is regarded as a highly salt tolerant halophyte crop, of great potential for cultivation on saline areas around the world. Fourteen quinoa genotypes of different geographical origin, differing in salinity tolerance, were grown under greenhouse conditions. Salinity treatment started on 10 day old seedlings. Six weeks after the treatment commenced, leaf sap Na and K content and osmolality, stomatal density, chlorophyll fluorescence characteristics, and xylem sap Na and K composition were measured. Responses to salinity differed greatly among the varieties. All cultivars had substantially increased K⁺ concentrations in the leaf sap, but the most tolerant cultivars had lower xylem Na⁺ content at the time of sampling. Most tolerant cultivars had lowest leaf sap osmolality. All varieties reduced stomata density when grown under saline conditions. All varieties clustered into two groups (includers and excluders) depending on their strategy of handling Na⁺ under saline conditions. Under control (non-saline) conditions, a strong positive correlation was observed between salinity tolerance and plants ability to accumulate Na⁺ in the shoot. Increased leaf sap K⁺, controlled Na⁺ loading to the xylem, and reduced stomata density are important physiological traits contributing to genotypic differences in salinity tolerance in quinoa, a halophyte species from Chenopodium family.     

Biochemical characterization of maize (Zea mays L.) for salt tolerance

The inherent differences for salt tolerance in two maize cultivars (Agatti-2002 and Sahiwal-2002) were evaluated in pot experiments. Plants were grown in half-strength of Hoagland nutrient solution added with 0, 80, 100, 120, 140 and 160 mM of NaCl. Salt stress markedly reduced the shoot and root lengths and fresh and dry masses. Reduction in growth attributes was more pronounced in cv. Agatti-2002 than cv. Sahiwal-2002. Both maize cultivars exhibited significant perturbations in important biochemical attributes being employed for screening the crops for salt tolerance. Cultivar Sahiwal-2002 was found salt tolerant as compared to cv. Agatti-2002 because it exhibited lower levels of H₂O₂, malondialdehyde (MDA) and higher activities of antioxidant enzymes. In addition, cultivar Sahiwal-2002 exhibited less salt-induced degradation of photosynthetic pigments, lower levels of toxic Na⁺ and Cl^?  and higher endogenous levels of K⁺ and K⁺/Na⁺ ratio. The results indicate that salt stress induced a marked increase in MDA, H₂O₂, relative membrane permeability, total soluble proteins and activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase andascorbate peroxidase). Moreover, increase in endogenous levels of Na⁺ and Cl^?  and decrease in K⁺ and K⁺/Na⁺ ratio and photosynthetic pigments were recorded in plants grown under salinity regimes.     

Exogenously applied paclobutrazol modulates growth in salt-stressed wheat plants

Effect of paclobutrazol (PBZ) treatment on salinity tolerance of wheat (Triticum aestivum) was investigated on a salt-tolerant (Karchia-65) and salt-sensitive (Ghods) cultivars. Salinity significantly reduced the investigated growth parameters such as plant height, length and area of sixth leaf, root length, fresh and dry weight of shoot, roots and sixth leaf, water content (WC) of plant and seeds weight in the both cultivars. The negative effect of salinity in Ghods cultivar was more than Karchia cultivar. However, PBZ treatment reduced the growth in both cultivars, the differences in plant growth among various levels of NaCl decreased in PBZ-treated plants. Salt stress resulted in high accumulation of Na⁺ in the sixth leaf and roots in both cultivars, particularly in Ghods cultivar. Against Karchia cultivar, salt stress decreased the storage of K⁺, P and N in sixth leaf and roots in Ghods cultivar. In the both cultivars, PBZ treatment enhanced the K⁺, P and N contents in sixth leaf and roots by increasing salinity. Although PBZ treatment decreased the growth of plants, it improved the weight of seeds against stress damage. PBZ treatment reduced the accumulation of harmful Na⁺ ion in plant tissues while increased the K⁺, P and N contents. These observations suggest that PBZ treatment may increase tolerance by diminishing ionic imbalance caused by salt stress.     

短期NaCl胁迫对不同小麦品种幼苗K+吸收和Na+、K+积累的影响

为研究盐胁迫下小麦幼苗生长及Na⁺、K⁺的吸收和积累规律,以中国春、洲元9369和长武134等3种耐盐性不同小麦品种为材料,采用非损伤微测技术检测盐胁迫2 d后的根系K⁺离子流变化,并对植株体内的Na⁺、K⁺含量进行测定。结果表明:短期(2d)盐胁迫对小麦生长有抑制作用,且对根系的抑制大于地上部,耐盐品种下降幅度小于盐敏感品种。盐胁迫下,小麦根际的 K⁺大量外流,盐敏感品种中国春K⁺流速显著高于耐盐品种长武134,最高可达15倍。小麦幼苗地上部分和根系均表现为Na⁺积累增加,K⁺积累减少,Na⁺/K⁺比随盐浓度增加而上升。中国春限Na⁺能力显著低于长武134,Na⁺/K⁺则显著高于长武134。综上所述,盐胁迫下造成小麦组织器官中Na⁺/K⁺比上升的主要原因是根系K⁺大量外流和Na⁺的过量积累,耐盐性不同的小麦品种间差异显著,并认为根系对K⁺的保有能力可能是作物耐盐性评价的一个重要指标。     

Physiological and biochemical parameters for evaluation and clustering of rice cultivars differing in salt tolerance at seedling stage

Salinity tolerance levels and physiological changes were evaluated for twelve rice cultivars, including four white rice and eight black glutinous rice cultivars, during their seedling stage in response to salinity stress at 100 mM NaCl. All the rice cultivars evaluated showed an apparent decrease in growth characteristics and chlorophyll accumulation under salinity stress. By contrast an increase in proline, hydrogen peroxide, peroxidase (POX) activity and anthocyanins were observed for all cultivars. The K⁺/Na⁺ ratios evaluated for all rice cultivars were noted to be highly correlated with the salinity scores thus indicating that the K⁺/Na⁺ ratio serves as a reliable indicator of salt stress tolerance in rice. Principal component analysis (PCA) based on physiological salt tolerance indexes could clearly distinguish rice cultivars into 4 salt tolerance clusters. Noteworthy, in comparison to the salt-sensitive ones, rice cultivars that possessed higher degrees of salt tolerance displayed more enhanced activity of catalase (CAT), a smaller increase in anthocyanin, hydrogen peroxide and proline content but a smaller drop in the K⁺/Na⁺ ratio and chlorophyll accumulation.     

Osmolyte accumulation in different rape genotypes under sodium chloride salinity

Physiological mechanisms of two rape (Brassica napus L.) genotype adaptation to chlorine salinity were investigated. The plants of two cultivars (Olga and Westar) differing in salt tolerance were grown in the pots filled with Perlite on the Hoagland and Snyder’s medium under controlled conditions. At a stage of 3–4 true leaves, the plants experienced 7-day-long salinity induced by a single addition of NaCl to the nutrient medium in order to attain desired final salt concentration (from 50 to 400 mM). The obtained results showed that a greater salt tolerance of cv. Olga plants (as compared with cv. Westar) could be accounted for by a capability of their root cells to uptake water under high salinity (300–400 mM NaCl), which is evident from a greater content of water in the tissues of cv. Olga. This was ensured by a sharp fall of the osmotic potential of the cellular contents (down to −2.3 MPa) at a low water potential of nutrient solution owing to more active uptake of Na⁺ (57–61 μeq/g fr wt) and K⁺ (210–270 μeq/g fr wt) as well as active accumulation of proline (30–50 μmol/g fr wt). The latter is caused by a reduced activity of proline dehydrogenase and retarded degradation of this osmolyte. It is important that, in contrast to less tolerant genotype, the rape plants of salt-resistant cultivar were able to maintain the K⁺/Na⁺ ratio at a rather high level at salinity of different degree, which made it possible to preserve ionic homeostasis under adverse conditions. Original Russian Text ? A.M. Mokhamed, G.N. Raldugina, V.P. Kholodova, Vl.V. Kuznetsov, 2006, published in Fiziologiya Rastenii, 2006, Vol. 53, No. 5, pp. 732–738.     

Effects of salinity stress on some growth,physiological, biochemical parameters and nutrients in two pistachio (Pistacia vera L.) rootstocks

In the present study, effects of salinity stress were evaluated in the leaves and roots of two pistachio cultivars (Badami-Rize-Zarand (BZ) and Badami-e-Sefid (BS)). In overall, salinity negatively affects growth of both cultivars with more pronounced effects on BS. The physiological reason of the reduction could be attributed to some extent to more depletion of photosynthetic pigment in BS. In both cultivars, salinity increased proline content. Moderate and high salinities increased the soluble sugar contents in BZ. In both cultivars, Na⁺ content increased in plant organs with increasing Na⁺ in the media. Salinity treatment decreased the Fe and Pi contents in BS cultivar, while they remained unchanged in BZ. These results show that BZ cultivar exhibits more tolerance to salinity stress than BS cultivar possibly by better growth performance, accumulating more osmolytes, lesser accumulation of toxic sodium ion and lower Na⁺/K⁺ in the shoots as well as maintaining nutrient contents.     

Mechanisms of salt tolerance and interactive effects of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> inoculation on maize cultivars grown under salt stress conditions

The present work has been performed to study the growth and metabolic activities of two maize cultivars (cv. 323 and cv. 324) which are shown to have different tolerances to salt stress and to determine the effects of inoculation with Azospirillum spp. Along with identifying the mechanisms of maize salt tolerance and the role of Azospirillum (growth promoting rhizobacteria) in elevating salinity stress conditions is examined Maize cv. 323 was the most sensitive to salinity, while cultivar 324 was the most resistant of the 12 maize cultivars tested. Cultivars differences were apparent with certain growth criteria as well as related metabolic activities. The lack of a negative response to increasing NaCl concentration for water content, dry matter yield and leaf area of cv. 324 up to a concentration of – 0.6 MPa indicated salt tolerance. While for cv. 323 there was a marked inhibitory effect of salinity on growth. In the tolerant cv. 324, soluble and total saccharides, soluble protein in shoots and total protein in roots increased with salinity stress. The sensitivity of cv. 323 however was associated with depletion in saccharides and proteins. Proline accumulation was higher and detected earlier at a lower salinity concentration in the salt sensitive cv. 323 comapred to the salt tolerant cv. 324. When salt stressed maize was inoculated with Azospirillum, proline concentration declined significantly. The present study showed, in general, that the concentration of most amino acid increased on exposure to NaCl as well as when inoculated with Azospirillum. The relatively high salt tolerance of cv. 324, compared with cv. 323 was associated with a significantly high K⁺/Na⁺ ratio. Azospirillum inoculation markedly altered the selectivity of Na⁺, K⁺ and Ca⁺⁺ especially in the salt sensitive cultivar cv. 323. Azospirillum restricted Na⁺ uptake and enhanced the uptake of K⁺ and Ca⁺⁺ in cv. 323. A sharp reduction in the activity of nitrate reductase and nitrogenase in shoots and roots of both cultivars was induced by salinity stress. This reduction in NR and NA activity was highly significant at all salinity concentrations. Azospirillum inoculation stimulated NR and nitrogenase activity in both shoots and roots of both cultivars. The differential effect of Azospirillum inoculation on maize cv. 323 and cv. 324 illustrates the different sensitivity of these two cultivars to stress, but still does not provide any clues as to the key events leading to this difference.     

Salt tolerance of maize (Zea mays L.): the role of sodium exclusion

The influence of NaCl and Na₂SO₄ on growth of two maize cultivars (Zea mays cv. Pioneer 3906 and cv. Across 8023) differing in Na⁺ uptake was investigated in two green-house experiments. Na⁺ treatment with different accompanying anions (Cl^?/SO₄^2?) showed that ion toxicity was caused by Na⁺. While shoot growth of the two cultivars was markedly affected by salt in comparison to the control during the first 2–3 weeks, there were only slight differences between the cultivars. The shoot Ca²⁺ concentration was reduced in both cultivars, and the youngest leaves contained an even lower concentration compared with the rest of the shoot. During this first phase, Across 8023 tended to have higher concentrations of Ca²⁺ than Pioneer 3906. The Na⁺-excluding cultivar Pioneer 3906 showed continuous, although reduced, growth compared with the control, while the Na⁺ concentration in the shoot decreased until flowering. Cultivar Across 8023 accumulated Na⁺ until flowering: the reduction in the growth of stressed plants was greater than that for Pioneer 3906. Leaves of cultivar Across 8023 showed clear toxic symptoms, while those of the more salt-tolerant cultivar Pioneer 3906 did not. It is concluded that Na⁺ exclusion contributes to the salt tolerance of maize.     

Physiological Characteristics and Alternative Respiratory Pathway under Salt Stress in Two Wheat Cultivars Differing in Salt Tolerance

Changes in respiratory pathway, dry weight, contents of proline, ATP, Na⁺and K⁺were investigated under five salinity treatments in the leaves of plants of spring wheat (Triticum aestivumL.). Two cultivars differing in salt resistance, namely, 89122 (salt-tolerant) and Longchun13 (salt-sensitive), were used. The decrease in dry weight and K⁺content was observed with the increasing NaCl concentrations, but more in cv. 89122 plants than in Longchun13 plants. Contents of proline and Na⁺in both cvs increased greatly, but the former increased more in 89122 while the latter more in Longchun13 plants. In all salinity treatments tested, a salt-induced increase in the activity of the alternative pathway was found, although cytochrome pathway (CP) still remained the main electron transport pathway. ATP production changed in parallel with CP operation. Cv. 89122 plants could produce more ATP than cv. Longchun13 plants exposed to each salinity treatment and their ATP generation could even be stimulated in contrast to its rapidly decline in Longchun13 plants with increased salinity stress. The possible relationship between respiration metabolism and above mentioned physiological changes is discussed.     

Effects of Salinity and Relative Humidity on Two Melon Cultivars Differing in Salt Tolerance

The effects of increasing relative humidity on the growth and salt tolerance of two melon (Cucumis melo L.) cultivars, Revigal C-8 (salt sensitive) and Galia (salt tolerant) was investigated. One month after germination, the plants were exposed for 15 d to 0 (control) and 80 mM NaCl, under relative humidity (RH), 30 and 70 %. The growth of the whole plant, leaf, stem and root of cv. Revigal C-8 was increased with increasing RH. On the other hand, cv. Galia showed an increase in root growth with increasing RH only under the NaCl treatment. Under salinity, most of the Na⁺ was withheld in the stems. An increase in RH in the NaCl treatment significantly decreased Na⁺ and Cl^– concentrations in leaves of cv. Revigal C-8, while it had no effect on their concentrations in cv. Galia. In both cultivars, increasing RH under NaCl condition significantly decreased water contents in leaves and stems, and increased osmotic potential in roots. The amount of the root exudate of cv. Galia was significantly decreased with increasing RH, while it was not affected in cv. Revigal C-8. Under the NaCl treatment, cv. Galia had significantly higher leaf osmotic potential than cv. Revigal C-8 at both relative humidities and higher amount of root exudate at 30 % RH.     

Guard cell cation channels are involved in Na+–induced stomatal closure in a halophyte

The halophyte Aster tripolium, unlike well-studied non-halophytic species, partially closes its stomata in response to high Na⁺ concentrations. Since A. tripolium possesses no specific morphological adaptation to salinity, this stomatal response, preventing excessive accumulation of Na⁺ within the shoot via control of the transpiration rate, is probably a principal feature of its salt tolerance within the shoot. The ionic basis of the stomatal response to Na⁺ was studied in guard cell protoplasts from A. tripolium and from a non-halophytic relative, Aster amellus, which exhibits classical stomatal opening on Na⁺. Patch-clamp studies revealed that plasma membrane K⁺ channels (inward and outward rectifiers) of the halophytic and the non-halophytic species are highly selective for K⁺ against Na⁺, and are very similar with respect to unitary conductance and direct sensitivity to Na⁺. On the other hand, both species possess a significant permeability to Na⁺ through non-rectifying cation channels activated by low (physiological) external Ca²⁺ concentrations. Finally, it appeared that the differential stomatal response between the two species is achieved, at least in part, by a Na⁺-sensing system in the halophyte which downregulates K⁺ uptake. Thus, increases in guard cell cytosolic Na⁺ concentration in A. tripolium but not in A. amellus, lead to a delayed (20–30 min) and dramatic deactivation of the K⁺ inward rectifier. This deactivation is probably mediated by an increase in cytosolic Ca²⁺ since buffering it abolishes the response. The possible role of K⁺ inward rectifiers in the response of A. tripolium’s stomata to Na⁺, suggested by patch-clamp studies, was confirmed by experiments demonstrating that specific blockade of inward rectifying channels mimics Na⁺ effects on stomatal aperture, and renders aperture refractory to Na⁺.     

Growth and ion relations in response to combined salinity and waterlogging in the perennial forage legumes Lotus corniculatus and Lotus tenuis

Lotus tenuis (Wadst. & Kit.) is a perennial legume widely grown for pasture in the flood-prone and salt affected Pampa region of Argentina. The physiology of salt and waterlogging tolerance in L. tenuis (four cultivars) was evaluated, and compared with Lotus corniculatus (three cultivars); the most widely cultivated Lotus species. Overall, L. tenuis cultivars accumulated less Na⁺ and Cl⁻, and more K⁺ in shoots than L. corniculatus cultivars, when exposed to 200 mM NaCl for 28 days in aerated or in stagnant solutions. Root porosity was higher in L. tenuis cultivars due to greater aerenchyma formation. In a NaCl dose–response experiment (0–400 mM NaCl in aerated solution), L. tenuis (cv. Chaja) accumulated half as much Cl⁻ in its shoots than L. corniculatus (cv. San Gabriel) at all external NaCl concentrations, and about 30% less shoot Na⁺ in treatments above 250 mM NaCl. Ion distributions in shoots were determined for plants at 200 mM NaCl. L. tenuis (cv. Chaja) again accumulated about half as much Cl⁻ in old leaves, young leaves and stems, compared with concentrations in L. corniculatus (cv. San Gabriel). There were not, however, significant differences between the two species for Na⁺ concentrations in the various shoot tissues. The higher root porosity, and maintenance of lower shoot Cl⁻ and Na⁺ concentrations in L. tenuis, compared with L. corniculatus, contributes to the greater tolerance to combined salt and waterlogging stress in L. tenuis. Moreover, significant variation for tolerance to combined salinity and waterlogging stress was identified within both L. tenuis and L. corniculatus.     

Control of xylem Na+ loading and transport to the shoot in rice and barley as a determinant of differential salinity stress tolerance

Control of xylem Na⁺ loading has often been named as the essential component of salinity tolerance mechanism. However, it is less clear to what extent the difference in this trait may determine differential salinity tolerance between species. In this study, barley (Hordeum vulgare L. cv. CM72) and rice (Oryza sativa L. cv. Dongjin) plants were grown under two levels of salinity. Na⁺ and K⁺ concentrations in the xylem sap, and shoot and root tissues were measured at different time points after stress onset. Salt‐exposed rice plants prevented xylem Na⁺ loading for several days, but failed to control this process in the longer term, ultimately resulting in a massive Na⁺ shoot loading. Barley plants quickly increased xylem Na⁺ concentration and its delivery to the shoot (most likely for the purpose of osmotic adjustment) but were able to reduce this process later on, keeping most of accumulated Na⁺ in the root, thus maintaining non‐toxic shoot Na⁺ level. Rice plants increased shoot K⁺ concentration, while barley plants maintained higher root K⁺ concentration. Control of xylem Na⁺ loading is remarkably different between rice and barley; this difference may differentiate the extent of the salinity tolerance between species. This trait should be investigated in more detail to be used in the breeding programs aimed to improve salinity tolerance in crops.     

Vegetative growth,compatible solute accumulation,ion partitioning and chlorophyll fluorescence of ‘Malas-e-Saveh’ and ‘Shishe-Kab’ pomegranates in response to salinity stress

The present research was conducted to assess physiological responses of ‘Malas-e-Saveh’ (Malas) and ‘Shishe-Kab’ (Shishe) pomegranates to water of different salt content and electrical conductivity (1.05, 4.61, and 7.46 dS m^?1). Both cultivars showed a reduced trunk length due to salinity. Relative water content and stomatal conductivity of both cultivars were significantly reduced under salt stress, but ion leakage increased. In both cultivars, total chlorophyll (Chl) and carbohydrates decreased with rise in salinity, while proline accumulation increased. With salinity increment, the Chl fluorescence parameters (maximum photochemical efficiency of PSII and effective quantum yield of PSII) declined significantly in both cultivars, with higher reduction observed in Shishe. Generally, more Na⁺ accumulated in shoots and more Cl^? was observed in leaves. Cl^? accumulation increased by salinity in leaves of Malas, but it was reduced in Shishe. The K⁺/Na⁺ ratio in leaves decreased in both cultivars by salinity increment. Malas was less affected by osmotic effects of NaCl, but it accumulated more Cl^? in its leaves. Thus, Malas might be more affected by negative effects of salinity.     

The response of Mo-hydroxylases and abscisic acid to salinity in wheat genotypes with differing salt tolerances

The differential responses of the wheat cultivars Shi4185 and Yumai47 to salinity were studied. The higher sensitivity of Yumai47 to salinity was linked to a greater growth reduction under salt stress, compared to more salt-tolerant Shi4185. Salinity increased the Na⁺, proline and superoxide anion radical (O₂ ^?) contents in both cultivars. Leaf Na⁺ content increased less in the more salt-tolerant cultivar Shi4185 than salt-sensitive Yumai47. The proline content increased more significantly in Shi4185 than Yumai47; on the contrary, superoxide anion radical content increased less in Shi4185 than Yumai47. This data indicated that wheat salinity tolerance can be increased by controlling Na⁺ transport from the root to shoot, associated with higher osmotic adjustment capability and antioxidant activity. Although salinity increased aldehyde oxidase (AO) activity and abscisic acid (ABA) content in the leaves and roots of both cultivars following the addition of NaCl to the growth medium, AO and ABA increased more in the salt-sensitive cultivar Yumai47 than the more salt-tolerant cultivar Shi4185. Xanthine dehydrogenase (XDH) activity in the leaves of both cultivars increased with increasing concentrations of NaCl; however, leaf XDH activity increased more significantly in Yumai47 than Shi4185. Root XDH activity in Shi4185 decreased with increasing NaCl concentrations, whereas salinity induced an increased root XDH activity in Yumai47. The involvement of AO and XDH enzymatic activities and altered ABA content in the response mechanisms of wheat to salinity are discussed herein.