Expression of the Vigna aconitifolia P5CSF129A gene in transgenic pigeonpea enhances proline accumulation and salt tolerance

Abiotic stress is the major limiting factor of plant growth and crop yield which can be improved by osmoprotectants. Proline acts as an osmoprotectant and plays an important role in osmotic balancing, protection of sub-cellular structures, enzymes and in increasing cellular osmolarity that provide the turgor necessary for cell expansion under stress conditions. ?1-pyrroline-5-carboxylate synthetase (P5CS), a rate-limiting enzyme in proline biosynthesis which is known for conferring enhanced salt and drought stress is subjected to feedback inhibition by proline. Therefore, in the present study, we used a mutagenized version P5CSF129A of wild P5CS which is not subjected to feedback control. Efficient in vitro transformation of embryonic structures of pigeonpea (Cajanus cajan (L.) Millsp.) was obtained using Agrobacterium tumefaciens strain LBA4404 harbouring a modified binary vector pCAMBIA 1301 carrying the hptII gene for resistance to hygromycin sulphate, GUS reporter gene, encoding β-glucuronidase, and the Vigna aconitifolia P5CSF129A genes under a constitutive 35S promoter. Embryonic structures showed blue color when tested for GUS after first cycle of antibiotic selection. Integration of T-DNA into nuclear genome of transformed plants and its sexual transmission to the progeny of the transgenic plants are confirmed by PCR amplification of 340 bp hptII, 800 bp P5CSF129A fragments and Southern blot hybridization analysis. The resultant primary transgenic plants showed more proline accumulation than their non-transformed plants. Levels of proline were also elevated in T1 transgenic plants when grown in the presence of 200 mM NaCl. In addition to their enhanced growth performance, more chlorophyll and relative water content under high salinity, these plants also had lower levels of lipid peroxidation. This suggests that overproduction of proline might play an important role against salt shock and cellular integrity.     

拟南芥AtCIPK23基因对烟草抗旱能力的影响

为了探索拟南芥AtCIPK23基因对烟草耐旱能力的影响,对3个转AtCIPK23基因阳性纯合株系KA13、KA14和KA44与野生型烟草K326（对照）进行了自然干旱处理,测定离体叶片的失水速率、叶绿素含量、相对电导率、脯氨酸和可溶性糖含量,并分析了转基因及野生型材料对活性氧的清除能力,对活性氧清除基因NtSOD、NtCAT和NtAPX及干旱胁迫相关基因NtDREB、NtLEA5和NtCDPK2的表达量进行检测。结果表明：（1）转基因烟草离体叶片的失水速率明显低于K326;自然干旱7 d后,野生型K326出现了明显的干旱胁迫症状;干旱7 d进行复水后,转基因株系的复水存活率明显高于K326。（2）转基因株系中的叶绿素、脯氨酸及可溶性糖含量比K326显著提高,电导率则明显降低。（3）野生型烟草K326中H₂O₂的积累量明显高于3个转基因株系,转基因株系中ROS清除机制的3个关键基因NtSOD、NtCAT和NtAPX被诱导上调表达。（4）抗旱相关基因NtDREB、NtLEA5和NtCDPK2仅在转基因烟草中受干旱诱导。研究认为,AtCIPK23基因可能具有提高植物抗旱能力的功能。     

Enhanced proline accumulation and salt stress tolerance of transgenic <Emphasis Type="Italic">indica</Emphasis> rice by over-expressing <Emphasis Type="Italic">P5CSF129A</Emphasis> gene

Δ¹-pyrroline-5-carboxylate synthetase (P5CS) is a proline biosynthetic pathway enzyme and is known for conferring enhanced salt and drought stress in transgenics carrying this gene in a variety of plant species; however, the wild-type P5CS is subjected to feedback control. Therefore, in the present study, we used a mutagenized version of this osmoregulatory gene-P5CSF129A, which is not subjected to feedback control, for producing transgenic indica rice plants of cultivar Karjat-3 via Agrobacterium tumefaciens. We have used two types of explants for this purpose, namely mature embryo-derived callus and shoot apices. Various parameters for transformation were optimized including antibiotic concentration for selection, duration of cocultivation, addition of phenolic compound, and bacterial culture density. The resultant primary transgenic plants showed more enhanced proline accumulation than their non-transformed counterparts. This proline level was particularly enhanced in the transgenic plants of next generation (T₁) under 150 mM NaCl stress. The higher proline level shown by transgenic plants was associated with better biomass production and growth performance under salt stress and lower extent of lipid peroxidation, indicating that overproduction of proline may have a role in counteracting the negative effect of salt stress and higher maintenance of cellular integrity and basic physiological processes under stress.     

Drought tolerance and antioxidant enzymatic activity in transgenic ‘Swingle’ citrumelo plants over-accumulating proline

In this study we investigated the effects of the high endogenous proline level on water relations, gas exchange and antioxidant enzymatic activity in leaves of transgenic ‘Swingle’ citrumelo rootstocks transformed with the P5CSF129A gene coding for the key-enzyme for proline synthesis, under water deficit. Leaf total water, osmotic and pressure potentials, stomatal conductance, photosynthetic rates and xylem sap flow were evaluated in non-transformed control and transgenic plants during water deficit treatment. Malondialdehyde (MDA) content, catalase (CAT; EC 1.11.1.6), superoxide dismutase (SOD; EC 1.15.1.1) and ascorbate peroxidase (APX; EC 1.11.1.11) activities were quantified in leaves collected based on their total water potential, representing the following conditions: irrigated (Ψ_w = −1.3 MPa), moderate stress (Ψ_w = −2.3 to −2.5 MPa), severe stress (Ψ_w = −3.8 to −3.9 MPa) and recovery (24 h after re-irrigation: Ψ_w = −1.3 to −1.9 MPa). Osmotic adjustment was observed in transgenic plants until 11 days after withholding water, while pressure potential in non-transformed controls was close to zero after nine days of water deprivation. This superior maintenance of turgor pressure in leaves of transgenic plants led to higher stomatal conductance, photosynthetic and transpiration rates when compared to non-transgenic plants. Drought caused a significant decrease in APX and SOD activities in control plants, followed by an increase after re-watering. On the other hand, CAT was more active in control than in transgenic plants under irrigated condition and both stress levels. Our results suggest that transgenic plants were able to cope with water deficit better than non-transformed controls since the high endogenous proline level acted not only by mediating osmotic adjustment, but also by contributing to gas exchange parameters and ameliorating deleterious effects of drought-induced oxidative stress.     

Free proline accumulation in drought-stressed plants

Summary Free-proline accumulation was measured in leaves of intact wheat (Triticum vulgare L. cv. Kalyan Sona), plantago (Plantago ovata Forsk-Isabgool), papavar (Papaver somnifera L. Opium poppy) and mustard (Brassica juncea L. var. Varuna) grown in the field with low to high field water content and thus they were subjected to water stress. Leaf water deficit in percentage was used to determine the degree of stress at the time of proline anlysis.Free proline content was higher in mustard leaves as compared to wheat, plantago and papavar leaves. Water stress enhances the proline content but at same water deficit level the content differ in the leaves of the plants studied.     

Heterologous expression of <Emphasis Type="Italic">P5CS</Emphasis> gene in chickpea enhances salt tolerance without affecting yield

Vigna Δ¹-pyrroline-5-carboxylate synthetase (P5CS) cDNA was transferred to chickpea (Cicer arietinum L.) cultivar Annigeri via Agrobacterium tumefaciens mediated transformation. Following selection on hygromycin and regeneration, 60 hygromycin-resistant plants were recovered. Southern blot analysis of five fertile independent lines of T0 and T1 generation revealed single and multiple insertions of the transgene. RT-PCR and Western blot analysis of T0 and T1 progeny demonstrated that the P5CS gene is expressed and produced functional protein in chickpea. T1 transgenic lines accumulated higher amount of proline under 250 mM NaCl compared to untransformed controls. Higher accumulation of Na⁺ was noticed in the older leaves but negligible accumulation in seeds of T1 transgenic lines as compared to the controls. Chlorophyll stability and electrolyte leakage indicated that proline overproduction helps in alleviating salt stress in transgenic chickpea plants. The T1 transgenics lines were grown to maturity and set normal viable seeds under continuous salinity stress (250 mM) without any reduction in plant yield in terms of seed mass.     

Two ecologically distinct subspecies ofHypochaeris radicata L.

Summary Two subspecies ofHypochaeris radicata were compared with respect to differences in drought tolerance. The soil water content of the sites ofH. radicata ssp. ericetorum Van Soest was always lower than that ofH. radicata L. ssp.radicata throughout a great part of the growing season. Two water culture experiments were conducted at different light intensities. Water stress was induced by addition of NaCl to the culture solution. Both subspecies accumulated free proline andmyo-inositol during water stress. The results are compared with those of field observations. In all experiments with stress application ssp.radicata showed heavier wilting symptoms than ssp.ericetorum, concomittantly with a lower osmotic potential of the cell sap, a higher percentage of dry-weight and irreversible desiccation of older leaves in some experiments after stress application. The observed effects are attributed to the higher transpiration rate maintained by ssp.radicata during stress. Free proline accumulation depended on the severity of the internal stress rather than on the applied level of external stress. At low light intensity the stress resulted in a significantly higher proline accumulation in ssp.ericetorum than in ssp.radicata whereas at high light intensity this was the reverse. No differences inmyo-inositol accumulation were observed in the water culture experiments. Since ssp.ericetorum occurs in a nitrogen poor environment, the effect of nitrogen deprivation on accumulation of free proline andmyo-inositol was investigated. Both subspecies tended to accumulate less proline under such conditions especially ssp.radicata. Accumulation ofmyo-inositol was not favoured by nitrogen deprivation in the water culture experiments. Neither of the subspecies accumulated proline during the sampling period in the field presumably as a result of the wet summer. Leaves of whole plants collected in the field and subsequently subjected to water deprivation showed a high capacity to accumulate free proline. The level ofmyo-inositol in the field was higher in ssp.ericetorum than in either ssp.radicata or control plants in the water culture experiments. When the cytoplasmic volume is estimated as 10% of the total cell volume, free proline andmyo-inositol account for 44–69% of the osmotic potential. It is concluded that ssp.ericetorum is better adapted to the drier environment by its higher capacity to accumulate proline and reduce transpiration during stress. Grassland species research group, publication no41.     

Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na+/H+ antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum)

Agriculture productivity is severely affected by soil salinity. One possible mechanism by which plants could survive salt stress is to compartmentalize sodium ions away from the cytosol. In the present work, transgenic buckwheat plants overexpressing AtNHX1, a vacuolar Na⁺/H⁺ antiporter gene from Arabidopsis thaliana, were regenerated after transformation with Agrobacterium tumefaciens. These plants were able to grow, flower and accumulate more rutin in the presence of 200 mmol/l sodium chloride. Moreover, the content of important nutrients in buckwheat was not affected by the high salinity of the soil. These results demonstrated the potential value of these transgenic plants for agriculture use in saline soil.     

Differential responses of the enzymes involved in proline biosynthesis and degradation in drought tolerant and sensitive cotton genotypes during drought stress and recovery

The relative water content (RWC), free proline levels and the activities of enzymes involved in proline metabolism were studied in drought tolerant (Ca/H 680) and drought sensitive (Ca/H 148) genotypes of cotton (Gossypium hirsutum L.) during induction of water stress and posterior recovery. Water stress caused a significant increase in proline levels and P5CS activity in leaves of both tolerant and sensitive genotypes, whereas the activity of P5CR increased minimally and the activity of OAT remains unchanged. The activity of PDH decreased under drought stress in both the genotypes. The leaf of tolerant genotype maintained higher RWC, photosynthetic activity and proline levels, as well as higher P5CS and P5CR activities under water stress than that of drought sensitive genotype. The drought induced proline levels and activities of P5CS and P5CR declined and tend to be equal to their respective controls, during recovery, whereas the PDH activity tends to increase. These results indicate that induction of proline levels by up regulation of P5CS and down regulation of PDH may be involved in the development of drought tolerance in cotton.     

The Novel Use of a Combination of Sonication and Vacuum Infiltration in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated Transformation of Kidney Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) with <Emphasis Type="Italic">lea</Emphasis> Gene

An efficient gene transfer system without tissue culture steps was developed for kidney bean by using sonication and vacuum infiltration assisted, Agrobacterium-mediated transformation. Transgenic kidney bean with a group 3 lea (late embryogenesis abundant) protein gene from Brassica napus was produced through this approach. Among 18 combinations of transformation methods, Agrobacterium-mediated transformation combined with 5 min sonication and 5 min vacuum infiltration turned to be optimal, resulting in the highest transformation efficiency. Transgenic kidney bean plants demonstrated enhanced growth ability under salt and water deficit stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by drought stress. Transgenic lines with high level of lea gene expression showed higher stress tolerance than lines with lower expression level. Stress tolerance of transgenic kidney bean correlated much better with lea gene expression levels than with gene integration results. There is no prior report on the production of transgenic kidney bean using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation.     

Transformation of radish (Raphanus sativus L.) via sonication and vacuum infiltration of germinated seeds with Agrobacterium harboring a group 3 LEA gene from B. napus

A protocol for producing transgenic radish (Raphanus sativus) was obtained by using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation. The Agrobacterium strain LBA4404 contained the binary vector pBI121-LEA (late embyogenesis abundant), which carried a Group 3 LEA gene, from Brassica napus. Among six combinations, Agrobacterium-mediated transformation assisted by a combination of 5-min sonication with 5-min vacuum infiltration resulted in the highest transformation frequency. The existence, integration and expression of transferred LEA gene in transgenic T₁ plants were confirmed by PCR, genomic Southern and Western blot analysis. Transgenic radish demonstrated better growth performance than non-transformed control plants under osmotic and salt stress conditions. Accumulation of Group 3 LEA protein in the vegetative tissue of transgenic radish conferred increased tolerance to water deficit and salt stress.     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na⁺/H⁺反向转运蛋白基因AtNHX1转入荞麦中,在2.0mg/L 6-BA、0.1mg/L IAA、1mg/L KT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株（转化频率为4.17%）。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na⁺及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K⁺的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na⁺/H⁺反向转运蛋白基因AtNHX1转入荞麦中,在2.0mg/L 6-BA、0.1mg/L IAA、1mg/L KT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株（转化频率为4.17%）。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na⁺及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K⁺的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

半干旱区城市环境下油松林分蒸腾特征及其影响因子

在城市环境下,由于不透水地面面积的增加,土壤-植物-大气之间水汽循环减弱,水汽调节能力差,因而研究城市树木蒸腾对环境因子的响应对于城市进行合理的水汽调节具有重要意义。于2017年生长季,在内蒙古呼和浩特市区树木园内选择58年生油松(Pinus tabulaeformis Carr.)作为研究树种,采用热扩散法测定其树干液流,并同步监测气象因子和土壤含水量变化,利用彭曼公式计算冠层气孔导度。结果表明:(1)生长季内,油松林分蒸腾存在明显日、月变化,晴天天气下林分蒸腾日变化呈单峰曲线,月林分蒸腾量5月最大,其次是7月、8月、6月和9月,分别为20.96、19.89、18.09、17.25 mm和7.49 mm。(2)油松林分蒸腾与饱和水汽压差、太阳总辐射、土壤含水量和风速均存在极显著相关关系(P<0.01),太阳总辐射、饱和水汽压差和土壤含水量是影响林分蒸腾的主要环境因子(R²=0.47、R²=0.31和R²=0.16),风速对林分蒸腾的影响程度最小(R²=0.12);不同降雨量对林分蒸腾的影响作...     

Deficit and excess of soil water impact on plant growth of Lotus tenuis by affecting nutrient uptake and arbuscular mycorrhizal symbiosis

The impact of deficit and excess of soil water on plant growth, morphological plant features, N and P plant nutrition, soil properties, Rhizobium nodulation and the symbiosis between arbuscular mycorrhizal (AM) fungi and Lotus tenuis Waldst. & Kit. were studied in a saline-sodic soil. Water excess treatment decreased root growth by 36% and increased shoot growth by 13% whereas water deficit treatment decreased both root and shoot growth (26 and 32%, respectively). Differences between stress conditions on shoot growth were due to the ability of L. tenuis to tolerate low oxygen concentration in the soil and the sufficiency of nutrients in soil to sustain shoot growth demands. Water excess treatment decreased pH, and increased available P and labile C in soil. Water deficit treatment decreased available P and also increased labile C. In general, N and P acquisition were affected more by water excess than water deficit. The number of nodules per gram of fresh roots only increased in water excess roots (97%). Under both stress conditions there was a significant proportion of roots colonized by AM fungi. Compared to control treatment, arbuscule formation decreased by 55 and 14% under water excess and water deficit, respectively. Vesicle formation increased 256% in water excess treatment and did not change under water deficit treatment. L. tenuis plants subjected to water deficit or excess treatments could grow, nodulated and maintained a symbiotic association with AM fungi by different strategies. Under water excess, L. tenuis plants decreased root growth and increased shoot growth to facilitate water elimination by transpiration. Under water deficit, L. tenuis plants decreased root growth but also shoot growth which in turn significant decreased the shoot/root ratio. In the present study, under water excess conditions AM fungi reduced nutrient transfer structures (arbuscules), the number of entry points and spore, and hyphal densities in soil, but increased resistance structures (vesicles). At water deficit, however, AM fungi reduced external hyphae and arbuscules to some extent, investing more in maintaining a similar proportion of vesicles in roots and spores in soil compared to control treatment.     

Effect of water stress on growth, osmotic adjustment, cell wall elasticity and water-use efficiency in Spartina alterniflora

In the northern spring–summer season of 2004–2005, vegetative propagated plants of Spartina alterniflora were grown under control and water stress conditions on the Mediterranean sea shore of the south-east of Tunis. Control plants were irrigated every week and water stress plants were irrigated until the soil achieved 50% (mild stress) and 25% (severe stress) field capacity (FC). Dry and fresh weight at the whole plant level (g plant⁻¹), shoot to root ratio on dry and fresh weight, photosynthesis (A), transpiration rate (E), instantaneous water-use efficiency (WUE_i), leaf water potential (Ψw), leaf water content (WC), osmotic potential at full turgor (Ψs¹⁰⁰), osmotic potential at turgor loss point (Ψs⁰), osmotic adjustment (OA), proline, sugars, inorganic compounds and cell wall elasticity (CWE) were evaluated during a period of 6 days period between 82 and 90 days after the beginning of treatment (DAT). Plants grown under severe and mild-water stress showed lower Ψw than in control plants with values that averaged −3.1, −1.6 and −0.9 MPa, respectively. S. alterniflora plants submitted to mild-water stress exhibited OA and a decrease in CWE. However, under severe water stress the OA was not observed and CWE also decreased, but it was higher than in the mild-water stress. OA was mainly explained by the accumulation of nitrates, sugars and at a lesser degree, proline. S. alterniflora had a strong decline of the dry and fresh weight of the whole plant associated to a marked decrease of photosynthesis (A) and transpiration (E) in response to water stress, although WUE_i was increased. These results suggest that OA and WUE_i can be important components of the water stress adaptation mechanism in this species, but they are not sufficient enough to contribute to resistance to water stress.     

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na⁺ and K⁺ accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.     

Organogenesis and <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Eucalyptus saligna</Emphasis> with <Emphasis Type="Italic">P5CS</Emphasis> gene

The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ¹-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied. The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots was similar in both types of plants.