Overexpression of the Zygophyllum xanthoxylum Aquaporin, ZxPIP1;3, Promotes Plant Growth and Stress Tolerance.

Drought and salinity can result in cell dehydration and water unbalance in plants, which seriously diminish plant growth and development. Cellular water homeostasis maintained by aquaporin is one of the important strategies for plants to cope with these two stresses. In this study, a stress-induced aquaporin, ZxPIP1;3, belonging to the PIP1 subgroup, was identified from the succulent xerophyte Zygophyllum xanthoxylum. The subcellular localization showed that ZxPIP1;3-GFP was located in the plasma membrane. The overexpression of ZxPIP1;3 in Arabidopsis prompted plant growth under favorable condition. In addition, it also conferred salt and drought tolerance with better water status as well as less ion toxicity and membrane injury, which led to more efficient photosynthesis and improved growth vigor via inducing stress-related responsive genes. This study reveals the molecular mechanisms of xerophytes' stress tolerance and provides a valuable candidate that could be used in genetic engineering to improve crop growth and stress tolerance.

OPDA and ABA accumulation in Pb-stressed Zygophyllum fabago can be primed by salicylic acid and coincides with organ-specific differences in accumulation of phenolics.

Salicylic acid (SA) is a well-known priming agent that is widely used to protect plants against stressing agents, including heavy metals as Pb. A better understanding of the mechanisms that enable plants to counteract Pb toxicity would help to select strategies for land reclamation programs. Here we used a metallicolous population of Zygophyllum fabago to assess the extent to which SA pretreatment modulates Pb-induced changes in phenol metabolism and stress-related phytohormone levels in roots and leaves. Our data revealed that accumulation of different phytohormones, lignin, soluble and wall-bound phenolics as well as peroxidase (PRX) activity in Pb-stressed plants differed after SA-pretreatment. Exposure to Pb led to the induction of soluble and cell wall-bound PRX activities, particularly those involved in the oxidation of coniferyl alcohol and ferulic acid, while pretreatment with SA reduced the Pb-induced stimulation of PRX activities in roots but increased them in leaves. SA-treatment by itself induced accumulation of ABA and the JA-precursor 12-oxo-phytodienoic acid (OPDA) in the roots. Pb in turn inhibited these SA-induced effects with the exception of OPDA accumulation that was primed by the pretreatment. The SA treatment also induced accumulation of OPDA in leaves but suppressed the accumulation of JA-Ile although with relatively small absolute changes. Notably, Pb-induced accumulation of ABA was primed in the leaves of SA-pretreated plants. Together our data suggest that priming of OPDA accumulation in the roots and of ABA in the leaves by SA-pretreatment may play important regulatory roles, possibly via regulating PRX activities, for Pb stress in plants.

Different mechanisms of the metalliferous Zygophyllum fabago shoots and roots to cope with Pb toxicity.

Lead (Pb) remains classified as a priority pollutant. Zygophyllum fabago is considered an early colonizer of heavy metal-polluted soils under semiarid conditions, but physiological mechanisms underlying this colonizing capacity remain unclear. In order to characterize Z. fabago plants' performance on Pb-contaminated soils, we evaluated how Pb influenced root and shoot growth, carbon metabolism, and oxidative status. For that, 30-day-old seedlings from one population colonizing a mine tailing ("Mercader") at Murcia (southeast Spain) were exposed to 500-µM Pb(NO3)2 for 1 week. Results showed that this high dose of Pb induced no plant mortality nor senescence, though promoting plant nanism. Besides the efficiency of roots to accumulate Pb, shoots also demonstrate a high efficiency to translocate and accumulate this metal. Pb exposure decreased Zn uptake to the aerial part and reduced net photosynthetic rate (A), RuBisCO activity, chlorophyll, and soluble sugar contents in shoots. Moreover, in shoots, Pb exposure increased the levels of O2- and decreased antioxidant capacity, culminating with a loss of cell membrane integrity (electrolyte leakage) and increased protein oxidation. Compared to controls, exposed roots had less Mn and Zn levels, and despite the rise in H2O2 levels, they were able to modulate non-protein thiols presenting a robust defense capacity. This capacity may support the roots' ability to maintain cell membrane integrity (electrolyte leakage) with regard to control. Principal component analysis (PCA) contributed to elucidate how this species adjusts physiological mechanisms to cope with Pb toxicity, showing that roots and shoots evolved different antioxidant defenses, which demonstrates the importance of organ specificity in the response of Z. fabago to heavy metals.

ZxAKT1 is essential for K+ uptake and K+ /Na+ homeostasis in the succulent xerophyte Zygophyllum xanthoxylum.

The inward-rectifying K+ channel AKT1 constitutes an important pathway for K+ acquisition in plant roots. In glycophytes, excessive accumulation of Na+ is accompanied by K+ deficiency under salt stress. However, in the succulent xerophyte Zygophyllum xanthoxylum, which exhibits excellent adaptability to adverse environments, K+ concentration remains at a relatively constant level despite increased levels of Na+ under salinity and drought conditions. In this study, the contribution of ZxAKT1 to maintaining K+ and Na+ homeostasis in Z. xanthoxylum was investigated. Expression of ZxAKT1 rescued the K+ -uptake-defective phenotype of yeast strain CY162, suppressed the salt-sensitive phenotype of yeast strain G19, and complemented the low-K+ -sensitive phenotype of Arabidopsis akt1 mutant, indicating that ZxAKT1 functions as an inward-rectifying K+ channel. ZxAKT1 was predominantly expressed in roots, and was induced under high concentrations of either KCl or NaCl. By using RNA interference technique, we found that ZxAKT1-silenced plants exhibited stunted growth compared to wild-type Z. xanthoxylum. Further experiments showed that ZxAKT1-silenced plants exhibited a significant decline in net uptake of K+ and Na+ , resulting in decreased concentrations of K+ and Na+ , as compared to wild-type Z. xanthoxylum grown under 50 mm NaCl. Compared with wild-type, the expression levels of genes encoding several transporters/channels related to K+ /Na+ homeostasis, including ZxSKOR, ZxNHX, ZxSOS1 and ZxHKT1;1, were reduced in various tissues of a ZxAKT1-silenced line. These findings suggest that ZxAKT1 not only plays a crucial role in K+ uptake but also functions in modulating Na+ uptake and transport systems in Z. xanthoxylum, thereby affecting its normal growth.

Seasonal changes in antioxidative/oxidative profile of mining and non-mining populations of Syrian beancaper as determined by soil conditions.

Soil pollution by heavy metals/metalloids (HMMs) is a problem worldwide. To prevent dispersion of contaminated particles by erosion, the maintenance of a vegetative cover is needed. Successful plant establishment in multi-polluted soils can be hampered not only by HMM toxicities, but also by soil nutrient deficiencies and the co-occurrence of abiotic stresses. Some plant species are able to thrive under these multi-stress scenarios often linked to marked fluctuations in environmental factors. This study aimed to investigate the metabolic adjustments involved in Zygophyllum fabago acclimative responses to conditions prevailing in HMM-enriched mine-tailings piles, during Mediterranean spring and summer. To this end, fully expanded leaves, and rhizosphere soil, of three contrasting mining and non-mining populations of Z. fabago grown spontaneously in south-eastern Spain were sampled in two consecutive years. Approximately 50 biochemical, physiological and edaphic parameters were examined, including leaf redox components, primary and secondary metabolites, endogenous levels of salicylic acid, and physicochemical properties of soil (fertility parameters and total concentration of HMMs). Multivariate data analysis showed a clear distinction in antioxidative/oxidative profiles between and within the populations studied. Levels of chlorophylls, proteins and proline characterized control plants whereas antioxidant capacity and C- and S-based antioxidant compounds were biomarkers of mining plants. Seasonal variations were characterized by higher levels of alkaloids and PAL and soluble peroxidase activities in summer, and by soluble sugars and hydroxycinnamic acids in spring irrespective of the population considered. Although the antioxidant systems are subjected to seasonal variations, the way and the intensity with which every population changes its antioxidative/oxidative profile seem to be determined by soil conditions. In short, Z. fabago displays a high physiological plasticity that allow it to successfully shift its metabolism to withstand the multiple stresses that plants must cope with in mine tailings piles under Mediterranean climatic conditions.

Silicon improves photosynthesis and strengthens enzyme activities in the C3 succulent xerophyte Zygophyllum xanthoxylum under drought stress.

One main strategic adaptive mechanism adopted by succulent xerophyte species, resistance to drought stress is absorbing and accumulating large amounts of sodium (Na+) from poor and dry soil which was stored in photosynthesizing branches as well as leaves as major osmoregulators, while still accumulating and storing a great deal of silicon (Si) in roots to resist to arid environments. To understand the possible adaptive strategies underlying how Si accumulation stimulates growth and ameliorates the adverse environmental impacts of drought stress on the C3 succulent xerophyte Zygophyllum xanthoxylum, plants grown for 3 weeks were suffered different K2SiO3 concentrations (1.5-7.5mM) (3-15mM KCl as control) treatments in sand culture experiments. Plants were also treated with different osmotic stresses caused by polyethylene glycol (PEG 6000) and drought stress (maintain water content about 30% of field water capacity) (30% of FWC) with or without additional 2.5mMK2SiO3 (5mMKCl as control) treatment in sand culture and pot experiments, respectively. We found that 2.5mMK2SiO3 (5mMKCl as control) resulted in optimal plant growth and alleviated adverse influences of drought stress on Z. xanthoxylum, by strengthening the activities of superoxide dismutase, peroxidase and catalase, reducing membrane lipid peroxidation and decreasing soluble sugar and free proline concentrations, concomitantly, increasing tissue water content, leaf area and chlorophyll a concentration. The result of ion analysis indicated that the Si absorption of Z. xanthoxylum was markedly induced by drought stress and that the 2.5mMK2SiO3 (5mMKCl as control) treatment significantly increased the aboveground and root Si concentration under different osmotic stresses and 30% of field water capacity compared with the drought and drought with 5mMKCl treatments. Although the K+ concentration in root in the drought with 2.5mMK2SiO3 treatment was no significant changes compared with the drought treatment, K+ concentration in aboveground and root in drought with 2.5mMK2SiO3 treatment were significantly decreased by 42% and 65.2% compared with drought with 5mMKCl treatment under 30% of FWC, indicating that Si replaced the function of K+, thus stimulating the growth and mitigating adverse effects of Z. xanthoxylum under water deficit. These findings showed that the positive roles of Si in the drought tolerance of Z. xanthoxylum might be due to the ability of plant to accumulate a great quantity of Si and utilize it as an osmoregulator to copy with water deficit, which was coupled with an obvious improvement in photosynthetic activity and anti-oxidative enzyme activities.

Co-expression of tonoplast Cation/H(+) antiporter and H(+)-pyrophosphatase from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity, drought and field conditions.

Salinity and drought are major environmental factors limiting the growth and productivity of alfalfa worldwide as this economically important legume forage is sensitive to these kinds of abiotic stress. In this study, transgenic alfalfa lines expressing both tonoplast NXH and H(+)-PPase genes, ZxNHX and ZxVP1-1 from the xerophyte Zygophyllum xanthoxylum L., were produced via Agrobacterium tumefaciens-mediated transformation. Compared with wild-type (WT) plants, transgenic alfalfa plants co-expressing ZxNHX and ZxVP1-1 grew better with greater plant height and dry mass under normal or stress conditions (NaCl or water-deficit) in the greenhouse. The growth performance of transgenic alfalfa plants was associated with more Na(+), K(+) and Ca(2+) accumulation in leaves and roots, as a result of co-expression of ZxNHX and ZxVP1-1. Cation accumulation contributed to maintaining intracellular ions homoeostasis and osmoregulation of plants and thus conferred higher leaf relative water content and greater photosynthesis capacity in transgenic plants compared to WT when subjected to NaCl or water-deficit stress. Furthermore, the transgenic alfalfa co-expressing ZxNHX and ZxVP1-1 also grew faster than WT plants under field conditions, and most importantly, exhibited enhanced photosynthesis capacity by maintaining higher net photosynthetic rate, stomatal conductance, and water-use efficiency than WT plants. Our results indicate that co-expression of tonoplast NHX and H(+)-PPase genes from a xerophyte significantly improved the growth of alfalfa, and enhanced its tolerance to high salinity and drought. This study laid a solid basis for reclaiming and restoring saline and arid marginal lands as well as improving forage yield in northern China.

Profiling of rutin-mediated alleviation of cadmium-induced oxidative stress in Zygophyllum fabago.

Zygophyllum fabago grows in arid, saline soil, or disturbed sites, such as former industrial or mining areas. This species is able to grow in coarse mineral substrates contaminated with heavy metals. To investigate the effects of the flavonoid rutin (Rtn) on certain heavy metal stress responses such as antioxidant defense systems and water status, seedlings were subjected to 100 and 200 µM CdCl2 treatment without or with 0.25 and 1 mM Rtn for 7 and 14 d (days). Cd stress decreased growth (RGR), water content (RWC), leaf osmotic potential (Ψ(Π)), and chlorophyll fluorescence, all of which could be partly alleviated by addition of Rtn. Activities of superoxide dismutase, peroxidase (POX), ascorbate peroxidase, and glutathione reductase increased within the first 7 d after exposure to Cd. However, failure of antioxidant defense in the scavenging of reactive oxygen species (ROS) was evidenced by an abnormal rise in superoxide anion radical ( O2(•-)) and hydrogen peroxide contents and a decline in hydroxyl radical (OH(•)) scavenging activity, resulting in enhancement of lipid peroxidation (TBARS) as a marker of Cd-induced oxidative stress. However, exogenously applied Rtn considerably improved the stress tolerance of plants via a reduction in Cd accumulation, modulation of POX activity, increase of proline (Pro) content, decrease in TBARS and ROS content and consequent lowering of oxidative damage of membrane. Overall, 0.25 and 1 mM Rtn could protect Z. fabago from the harmful effects of 100 µM Cd-induced oxidative stress throughout the experiment.

Pb-induced responses in Zygophyllum fabago plants are organ-dependent and modulated by salicylic acid.

Zygophyllum fabago is a promising species for restoring heavy metal (HM) polluted soils, although the mechanisms involved in HM tolerance in this non-model plant remain largely unknown. This paper analyses the extent to which redox-active compounds and enzymatic antioxidants in roots, stems and leaves are responsible for Pb tolerance in a metallicolous ecotype of Z. fabago and the possible influence of salicylic acid (SA) pretreatment (24 h, 0.5 mM SA) in the response to Pb stress. SA pretreatment reduced both the accumulation of Pb in roots and even more so the concentration of Pb in aerial parts of the plants, although a similar drop in the content of chlorophylls and in the maximum quantum yield of photosystem II was observed in both Pb- and SA-Pb-treated plants. Pb increased the endogenous free SA levels in all organs and this response was enhanced in root tissues upon SA pretreatment. Generally, Pb induced a reduction in catalase, ascorbate peroxidase and glutathione reductase specific activities, whereas dehydroascorbate reductase was increased in all organs of control plants. SA pretreatment enhanced the Pb-induced H2O2 accumulation in roots by up-regulating Fe-superoxide dismutase isoenzymes. Under Pb stress, the GSH redox ratio remained highly reduced in all organs while the ascorbic acid redox ratio dropped in leaf tissues where a rise in lipid peroxidation products and electrolyte leakage was observed. Finally, an organ-dependent accumulation of proline and ß-carboline alkaloids was found, suggesting these nitrogen-redox-active compounds could play a role in the adaptation strategies of this species to Pb stress.

Sodium chloride improves photosynthesis and water status in the succulent xerophyte Zygophyllum xanthoxylum.

Zygophyllum xanthoxylum, a C(3) woody species, is a succulent xerophyte that is well adapted to arid environments. Our previous investigations showed that Na(+) has a positive effect on the growth of Z. xanthoxylum under drought conditions, which was closely related to high Na(+) accumulation in leaves. To reveal the physiological mechanisms underlying how Na(+) accumulation improves the drought resistance of Z. xanthoxylum, 3-week-old seedlings were treated with a series of additional external NaCl concentrations (5-150 mM) in sand culture experiments. Seedlings were also subjected to water deficit (30% of field water capacity) in the presence or absence of additional NaCl (50 mM) in pot experiments. The results indicated that 50 mM NaCl could mitigate deleterious impacts of water deficit on the growth of Z. xanthoxylum, by improving the relative water content, inducing a significant drop in leaf water potential and, concomitantly, increasing leaf turgor pressure and chlorophyll concentrations resulting in an enhancement of overall plant photosynthetic activity (i.e., photosynthetic rate and water use efficiency). Furthermore, NaCl (50 mM) could alleviate the inhibitory effect of water deficit on the activity of photosystem II in Z. xanthoxylum. The contribution of Na(+) to the total osmotic potential varied from 8% in the control to 13% in plants subjected to water deficit and, surprisingly, to 28% in plants grown in the presence of 50 mM NaCl under water deficit; however, the contribution of K(+) significantly decreased from 13 to 8%. These findings suggest that, under arid environments, Z. xanthoxylum is able to accumulate a high concentration of Na(+) in its leaves and use it directly for osmotic adjustment, which was coupled with an improvement in leaf hydration and photosynthetic activity.

The ZxNHX gene encoding tonoplast Na(+)/H(+) antiporter from the xerophyte Zygophyllum xanthoxylum plays important roles in response to salt and drought.

Sodium (Na(+)) has been found to play important roles in the adaptation of xerophytic species to drought conditions. The tonoplast Na(+)/H(+) antiporter (NHX) proved to be involved in the compartmentalization of Na(+) into vacuoles from the cytosol. In this study, a gene (ZxNHX) encoding tonoplast Na(+)/H(+) antiporter was isolated and characterized in Zygophyllum xanthoxylum, a succulent xerophyte growing in desert areas of northwest China. The results revealed that ZxNHX consisted of 532 amino acid residues with a conserved binding domain ((78)LFFIYLLPPI(87)) for amiloride and shared high similarity (73-81%) with the identified tonoplast Na(+)/H(+) antiporters in other plant species. Semi-quantitative RT-PCR analysis showed that the mRNA level of ZxNHX was significantly higher in the leaf than in stem or root. The transcript abundance of ZxNHX in Z. xanthoxylum subjected to salt (5-150 mM NaCl) or drought (50-15% of field water capacity (FWC)) was 1.4-8.4 times or 2.3-4.4 times that of plants grown in the absence of NaCl or 70% of FWC, respectively. Leaf Na(+) concentration in plants exposed to salt or drought was 1.7-5.2 times or 1.5-2.2 times that of corresponding control plants, respectively. It is clear that there is a positive correlation between up-regulation of ZxNHX and accumulation of Na(+) in Z. xanthoxylum exposed to salt or drought. Furthermore, Z. xanthoxylum accumulated larger amounts of Na(+) than K(+) in the leaf under drought conditions, even in low salt soil. In summary, our results suggest that ZxNHX encodes a tonoplast Na(+)/H(+) antiporter and plays important roles in Na(+) accumulation and homeostasis of Z. xanthoxylum under salt and drought conditions.

Amiloride reduces sodium transport and accumulation in the succulent xerophyte Zygophyllum xanthoxylum under salt conditions.

To investigate the effects of amiloride, which is an inhibitor of Na(+)/H(+) antiporter (NHX), on Na(+) accumulation and transport at the whole plant level, in this study, 3-week-old Zygophyllum xanthoxylum plants were exposed to 25, 50 and 100 mM NaCl with or without 0.5 mM amiloride. After 72 h of treatment, dry weight, tissue water content, Na(+) and K(+) concentrations, and transcript levels of ZxNHX were determined. The results indicated that, under salt conditions, leaf Na(+) concentration, total Na(+) quantity and root net Na(+) uptake rate in plants treated with amiloride are significantly lower than those in control plants. Amiloride remarkably increased Na(+) proportion in stem and decreased Na(+) proportion in leaf of Z. xanthoxylum. Furthermore, our results showed that the transcript levels of ZxNHX are down-regulated by amiloride. It is clear that the inhibition of vacuolar Na(+)/H(+) antiporter by amiloride could disrupt Na(+) accumulation of leaf, and reduce Na(+) uptake by root and Na(+) transport from stem to leaf, thus resulting in the growth inhibition of Z. xanthoxylum exposed to salt.

Impact of cadmium and zinc on growth and water status of Zygophyllum fabago in two contrasting metallicolous populations from SE Spain: comparison at whole plant and tissue level.

Cadmium and zinc accumulation and toxicity were assessed in whole plants and callus culture of two Zygophyllum fabago populations originating from two metallicolous sites in Murcia (southeast Spain), La Peña and Mazarrón, the first containing 2.8-times more Cd and five-times more Zn than the second. Seedlings from both ecotypes were exposed for 3 weeks to 1 or 10 µm Cd, and to 10 or 100 µm Zn in nutrient solution in a controlled environment. Calli from both ecotypes were exposed to 0.01, 0.1 or 1 mm Cd, and to 0.1, 1 or 5 mm Zn. Plants from both populations exhibited similar tolerance to Zn, while tolerance to Cd appeared more important in plants from La Peña than those from Mazarrón. Only minor differences were recorded in final Cd accumulation, with higher Cd retention in roots and stems of plants from La Peña. In both populations, transient decreases in the rate of Zn intake and translocation from root to shoot were recorded. This reduction in ion uptake was not more efficient for the population from the most contaminated area compared to the less contaminated area. Similar concentrations of Cd were found in cotyledon-derived calli from the two populations, but absorbed Cd induced conspicuous water stress in calli issues from Mazarrón but not in those from La Peña. It is concluded that, beside comparable levels of heavy metal concentration in tissues, the physiological strategy of tolerance may differ according to the metal and according to the considered population.

Heavy metal accumulation and tolerance in plants from mine tailings of the semiarid Cartagena-La Unión mining district (SE Spain).

Mine tailings are a characteristic of landscapes where mineral extraction has occurred. These tailings usually contain high heavy metal concentrations and have low fertility. In arid and semiarid zones, erosion may be an additional problem. The removal of these tailings is often impractical due to their large volumes. Therefore, a need exists to develop in situ low cost technologies to effect surface stabilization. The use of vegetation can be an attractive option, since there are some native plant species that can colonize parts of these polluted sites unaided. Some edaphic factors were investigated, including heavy metal concentrations, in three mine tailings from a semiarid mining zone in Southeast Spain. High total metal concentrations were found: 5000-8000 mg/kg for lead and 7600-12300 mg/kg for zinc. Two of these mine tailings had pH values between 6 and 7, while the other was acidic, with a pH of 2.5. Metal solubility was pH dependent, with more than 10% of the total zinc soluble in the acid substrate and less than 1% in the neutral substrates. The metal concentrations (copper, lead and zinc) in shoots of native vegetation colonizing in these sites were studied. No species of hyperaccumulators were found. The highest concentrations were found in Zygophyllum fabago, with 530 mg/kg zinc, Helichrysum decumbens with 390 mg/kg lead, and Tamarix sp. with 11 mg/kg copper. An analysis of the rhizospheric soil of these plants indicated that Lygeum spartum grew in pH 3 soil and had low metal concentration in shoots (40 mg/kg zinc and 41 mg/kg lead).