Aerenchyma Formation Research Articles

Adaptive roots of mangrove Avicennia marina: Structure and gene expressions analyses of pneumatophores

The Avicennia marina is a mangrove species widely distributed throughout the tropical and subtropical intertidal wetlands. To adapt to adverse tidal waves and hypoxia environments, A. marina has evolved a sophisticated root system to better secure itself on the muddy soil with downward-grown anchor roots and upward-grown aerial roots, called pneumatophores. However, the process behind the development of a negative-gravitropic pneumatophore is not understood. Paraffin sections reveal anatomical differences among the shoots, anchor roots, and gas exchanging pneumatophores, clearly reflecting their functional diversions. The pneumatophore, in particular, contains abundant aerenchyma tissues and a thin cap structure at the tip. Transcriptomic analyses of both anchor roots and pneumatophores were performed to elucidate gene expression dynamics during the formation of pneumatophores. The results show that the plant hormone auxin regulates multiple different root initiations. The auxin related gene IAA19 plays a key role in pneumatophore development while the interaction of ethylene and abscisic acid is important for aerenchyma formation. Moreover, the molecular mechanisms behind pneumatophore anti-gravitropic growth may be regulated by the reduced strength of the statolith formation signaling pathway. These results shed light on the mechanistic understanding of pneumatophore formation in mangrove plants.

A fast, efficient and high-throughput procedure involving laser microdissection and RT droplet digital PCR for tissue-specific expression profiling of rice roots

BackgroundIn rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues.ResultsWe optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database.ConclusionThe detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants.

Hypoxia-Responsive Class III Peroxidases in Maize Roots: Soluble and Membrane-Bound Isoenzymes

Flooding induces low-oxygen environments (hypoxia or anoxia) that lead to energy disruption and an imbalance of reactive oxygen species (ROS) production and scavenging enzymes in plants. The influence of hypoxia on roots of hydroponically grown maize (Zea mays L.) plants was investigated. Gene expression (RNA Seq and RT-qPCR) and proteome (LC–MS/MS and 2D-PAGE) analyses were used to determine the alterations in soluble and membrane-bound class III peroxidases under hypoxia. Gel-free peroxidase analyses of plasma membrane-bound proteins showed an increased abundance of ZmPrx03, ZmPrx24, ZmPrx81, and ZmPr85 in stressed samples. Furthermore, RT-qPCR analyses of the corresponding peroxidase genes revealed an increased expression. These peroxidases could be separated with 2D-PAGE and identified by mass spectrometry. An increased abundance of ZmPrx03 and ZmPrx85 was determined. Further peroxidases were identified in detergent-insoluble membranes. Co-regulation with a respiratory burst oxidase homolog (Rboh) and key enzymes of the phenylpropanoid pathway indicates a function of the peroxidases in membrane protection, aerenchyma formation, and cell wall remodeling under hypoxia. This hypothesis was supported by the following: (i) an elevated level of hydrogen peroxide and aerenchyma formation; (ii) an increased guaiacol peroxidase activity in membrane fractions of stressed samples, whereas a decrease was observed in soluble fractions; and (iii) alterations in lignified cells, cellulose, and suberin in root cross-sections.

Oxygen in the air and oxygen dissolved in the floodwater both sustain growth of aquatic adventitious roots in rice.

Flooding is an environmental stress that leads to a shortage of O2 that can be detrimental for plants. When flooded, deepwater rice grow floating adventitious roots to replace the dysfunctional soil-borne root system, but the features that ensure O2 supply and hence growth of aquatic roots have not been explored. We investigate the sources of O2 in aquatic adventitious roots and relate aerenchyma and barriers for gas diffusion to local O2 gradients, as measured by microsensor technology, to link O2 distribution in distinct root zones to their anatomical features. The mature root part receives O2 exclusively from the stem. It has aerenchyma that, together with suberin and lignin depositions at the water-root and cortex-stele interfaces, provides a path for longitudinal O2 movement toward the tip. The root tip has no diffusion barriers and receives O2 from the stem and floodwater, resulting in improved aeration of the root tip over mature tissues. Local formation of aerenchyma and diffusion barriers in the mature root channel O2 towards the tip which also obtains O2 from the floodwater. These features explain aeration of floating roots and their ability to grow under water.

Limited aerenchyma reduces oxygen diffusion and methane emission in paddy

Greenhouse gasses (GHG) emission from the agricultural lands is a serious threat to the environment. Plants such as rice (Oryza sativa L.) that are cultivated in submerged conditions (paddy field) contribute up to 19% of CH4 emission from agricultural lands. Such plants have evolved lysigenous aerenchyma in their root system which facilitates the exchange of O2 and GHG between aerial parts of plant and rhizosphere. Currently, the regulation of GHG and O2 via aerenchyma formation is poorly understood in plants, especially in rice. Here, a reverse genetic approach was employed to reduce the aerenchyma formation by analyzing two mutants i.e., oslsd1.1-m12 and oslsd1.1-m51 generated by Tos17 and T-DNA insertion. The wild-type (WT) and the mutants were grown in paddy (flooded), non-paddy and hydroponic system to assess phenotypic traits including O2 diffusion, GHG emission and aerenchyma formation. The mutants exhibited significant reductions in several morphophysiological traits including 20–60% aerenchyma formation at various distances from the root apex, 25% root development, 50% diffusion of O2 and 27–36% emission of methane (CH4) as compared to WT. The differential effects of the oslsd1.1 mutants in aerenchyma-mediated CH4 mitigation were also evident in the diversity of (pmoA, mcrA) methanotrophs in the rhizosphere. Our results indicate the novel pathway in which reduced aerenchyma in rice is responsible for the mitigation of CH4, diffusion of O2 and the root growth in rice. Limited aerenchyma mediated approach to mitigate GHG specially CH4 mitigation in agriculture is helpful technique for sustainable development.

Anatomical changes in stem and root of soybean plants submitted to salt stress.

The soybean is a legume that is widely cultivated in many countries due to the high levels of protein and oil contained in its seed, and is used for human and animal nutrition. However, salinity affects more than 800million hectares worldwide, limiting global agricultural production. The aim of this research was to evaluate the structural behaviour of the roots and stems under progressive salt stress, detailing the possible anatomical modifications to these organs in soybean plants during this stress. The plants were randomized into five treatments (0, 50, 100, 150 and 200mm NaCl). All the root regions studied and exposed to 100mm Na+ exhibited increases in the epidermis and endodermis and formation of lysogenic aerenchyma with increasing salinity, revealing the protective roles of these structures in reducing Na+ influx. In the stem, increases in the cortex and pith in the first internode subject to 100mm Na+ suggest anatomical responses that aim to minimize oxidative stress. Soybean plants subjected to progressive salt stress (>50mm Na+ ) avoided cavitation and loss of function linked to vessel elements, reducing the metaxylem in all the root and stem regions analysed. Finally, our results confirm anatomical changes to the roots and stems.

Structural and histochemical changes during in vitro development of Acrocomia aculeata (Arecaceae) seedlings

Embryo culture is important for the propagation of many species, and has proved to be efficient for overcoming seminal dormancy in Acrocomia aculeata, a neotropical palm having significant agro-industrial potential. Difficulties related to acclimatizing its in vitro cultured seedlings, however, restrict the commercial use of that technique. We therefore compared the development of A. aculeata seedlings cultivated ex vitro and in vitro to identify possible causes for its low propensity for acclimatization. Morphological, anatomical, micromorphometric, and histochemical evaluations were carried out on seedlings during both ex vitro and in vitro development. In vitro culture restricts the development of the haustorium, root system, and leaves. Restrictions of cell divisions in the epidermis and cell expansion in the parenchyma, together with the accumulation of phenolic compounds, lead to haustorium atrophy. The roots showed decreased thickness and branching, a reduced cortex, the absence of phenolic compound deposition, and early aerenchyma formation. The leaves show hypertrophied hypodermises, reduced chlorophyllous parenchyma, less carbohydrate deposition in the mesophyll, and less epicuticular wax. Those developmental limitations of the haustorium, roots, and leaves, contribute to the low acclimatization capacity of the seedlings. The incorporation into the culture medium of organic substances found in the endosperm, the hormonal stimulus of root development, as well as autotrophic culture may improve the acclimatization of A. aculeata seedlings. Acrocomia aculeata seedlings grown in vitro show haustorium atrophy, restrictions of root and mesophyll development, and limited depositions of epicuticular wax, characteristics that limit their acclimatization capacity.

Responses of physiological activities and lateral root anatomical structure of Cercis glabra to waterlogging stress

Cercis glabra is a colour-leaf tree with excellent ornamental value, whereas its physiological and morphological responses to waterlogging stress are still unclear. A potted study was conducted to determine the effects of waterlogging stress on antioxidative enzymes (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), lipid peroxidation (in terms of malondialdehyde (MDA) content), relative electric conductivity, and osmotic substance (free proline) of leaves and aerenchyma, lignification, suberization and Casparian strip of lateral roots of C. glabra. The result showed that the SOD, POD, and CAT activity and free proline content of C. glabra were significantly increased by the different degrees of waterlogging stress compared with the non-waterlogged treatment at 8 and 12 days, and the MDA content and relative electric conductivity of C. glabra leaves were significantly increased under the different degrees of waterlogging stress compared to the non-waterlogged treatment at 16 days, and the degrees of change increased among treatments was ranked as total waterlogged > semi-waterlogged > shallow waterlogged. The lateral roots of C. glabra not only formed developed aerenchyma in the cortex but also formed suberization and Casparian strip in the endodermis under semi-waterlogged treatment at 16 days. These results implied that C. glabra had a certain tolerance to waterlogging stress, which was associated with the increasing antioxidant enzyme activity and osmotic adjustment substance content, and with the formation of aerenchyma, suberization and Casparian strip in the lateral root to adapt to the waterlogged environment.

Keep Calm and Survive: Adaptation Strategies to Energy Crisis in Fruit Trees under Root Hypoxia.

Plants are permanently facing challenges imposed by the environment which, in the context of the current scenario of global climate change, implies a constant process of adaptation to survive and even, in the case of crops, at least maintain yield. O2 deficiency at the rhizosphere level, i.e., root hypoxia, is one of the factors with the greatest impact at whole-plant level. At cellular level, this O2 deficiency provokes a disturbance in the energy metabolism which has notable consequences on the yield of plant crops. In this sense, although several physiological studies describe processes involved in plant adaptation to root hypoxia in woody fruit trees, with emphasis on the negative impacts on photosynthetic rate, there are very few studies that include -omics strategies for specifically understanding these processes in the roots of such species. Through a de novo assembly approach, a comparative transcriptome study of waterlogged Prunus spp. genotypes contrasting in their tolerance to root hypoxia was revisited in order to gain a deeper insight into the reconfiguration of pivotal pathways involved in energy metabolism. This re-analysis describes the classically altered pathways seen in the roots of woody fruit trees under hypoxia, but also routes that link them to pathways involved with nitrogen assimilation and the maintenance of cytoplasmic pH and glycolytic flow. In addition, the effects of root hypoxia on the transcription of genes related to the mitochondrial oxidative phosphorylation system, responsible for providing adenosine triphosphate (ATP) to the cell, are discussed in terms of their roles in the energy balance, reactive oxygen species (ROS) metabolism and aerenchyma formation. This review compiles key findings that help to explain the trait of tolerance to root hypoxia in woody fruit species, giving special attention to their strategies for managing the energy crisis. Finally, research challenges addressing less-explored topics in recovery and stress memory in woody fruit trees are pointed out.

Genetic factors increasing barley grain yields under soil waterlogging

AbstractIn‐crop soil waterlogging can be caused by extreme rainfall events, high ground water tables, excessive irrigation, lateral ground water flow, either individually or in concert, and together these factors inhibit potential grain yields. However, the extent to which yield is influenced by the timing and duration of waterlogging relative to crop phenology is unknown. To investigate this, we conducted a range of waterlogging treatments on modern barley genotypes differing in their waterlogging tolerance, with tolerance conferred through aerenchyma formation under oxygen deficit conditions. Experiment 1 was conducted in a controlled environment using four waterlogging treatments: waterlogging at Zadoks stage (ZS) 12.5 for 1 or 2 months (WL1 and WL2, respectively), waterlogging at ZS 15 for 2 months (WL3), and waterlogging initiated 1 day before heading for 15 days (WL4). Experiment 2 was conducted in the field with WL2. Averaged across experiments, yield was reduced by 35% in W1 to 52% in WL3 due to fewer spikes/m2 and kernels/spike. WL4 had the greatest impact on yield (70% reduction) due to its effect on spikelet fertility and grain filling. Phenology was delayed 1–8 ZS at the end of waterlogging treatments, with the waterlogging‐susceptible cultivar Franklin showing the greatest delays, and waterlogging tolerant genotypes (Macquarie+, TAMF169) capable of aerenchyma formation under waterlogging having the least delays (0–4 ZS). Genotypes with aerenchyma formation QTL (Macquarie+) showed nonsignificant yield reduction compared with nonwaterlogged controls, preventing 23% yield loss under early phenological waterlogging stress. Late growth stage waterlogging substantially reduced average final grain yield by 70%.

Investigation of Hemicellulose Alteration in Fabaceae Root Cell Walls During Flooding-Induced Aerenchyma Formation

Investigation of Hemicellulose Alteration in Fabaceae Root Cell Walls During Flooding-Induced Aerenchyma Formation

Distance-to-Time Conversion Using Gompertz Model Reveals Age-Dependent Aerenchyma Formation in Rice Roots.

Distance-to-time conversion by using the Gompertz model compensates for the differences in root elongation rates among different genotypes and conditions, and reveals age-dependent aerenchyma formation.

Reactive oxygen species and reactive nitrogen species induce lysigenous aerenchyma formation through programmed cell death in rice roots under submergence

Nitric oxide (NO) plays a significant role in plants under different abiotic stress. However, the influence of NO and other reactive nitrogen species (RNS) in employing nitrosative stress in rice seedlings under submergence remains unexplored. Submergence also stimulates the development of lysigenous aerenchyma, facilitating oxygen supply to roots but the contribution of NO and RNS in aerenchyma formation under submergence is not known. Present study investigated the major components of the nitro-oxidative stress and their association with lysigenous aerenchyma development in the Sub1 near isogenic line of rice under submergence. Following submergence, Swarna showed increased NADPH oxidase (NOX) activity with excess reactive oxygen species (ROS) production in roots. Submergence also caused increased NO content and membrane lipid peroxidation in Swarna roots. Submergence-induced ROS and RNS accumulation in roots disturbed the redox homeostasis leading to the formation of lysigenous aerenchyma through programmed cell death (PCD). PCD was also accompanied by altered cytoplasmic streaming and DNA damage. In the present study Swarna Sub1 exhibited increased SOD, CAT, POX, APX, GR and GSNOR activity with subsequent detoxification of ROS and RNS; eventually decreasing the aerenchyma formation in root under submerged conditions. Overall, the study established ROS and RNS-mediated unique mechanism in lysigenous aerenchyma formation in rice roots under submergence.

Dynamics of radial oxygen loss in mangroves subjected to waterlogging.

Tidal flooding can directly result in oxygen (O2) shortage, however the functions of root aeration in flooding tolerance and O2 dynamics within mangroves are still poorly understood. Thus, in this study, the correlations among waterlogging tolerance, root porosity and O2 movement within the plants were investigated using two mangrove species (Aegiceras corniculatum and Bruguiera gymnorrhiza) and a semi-mangrove Heritiera littoralis. Based on the present data, the species A. corniculatum and B. gymnorrhiza, which possessed higher root porosity, exhibited higher waterlogging tolerance, while H. littoralis is intolerant. Increased root porosity, leaf stoma, and total ROL were observed in the roots of A. corniculatum and B. gymnorrhiza growing in stagnant solution when compared to respective aerated controls. As for ROL spatial pattern along roots, external anaerobic condition could promote ROL from apical root regions but reduce ROL from basal roots, leading to a 'tighter barrier'. In summary, the present study indicated that the plants (e.g., A. corniculatum and B. gymnorrhiza) prioritized to ensure O2 diffusion towards root tips under waterlogging by increasing aerenchyma formation and reducing O2 leakage at basal root regions.

A Role for Auxin in Ethylene-Dependent Inducible Aerenchyma Formation in Rice Roots.

Internal oxygen diffusion from shoot to root tips is enhanced by the formation of aerenchyma (gas space) in waterlogged soils. Lysigenous aerenchyma is created by programmed cell death and subsequent lysis of the root cortical cells. Rice (Oryza sativa) forms aerenchyma constitutively under aerobic conditions and increases its formation under oxygen-deficient conditions. Recently, we have demonstrated that constitutive aerenchyma formation is regulated by auxin signaling mediated by Auxin/indole-3-acetic acid protein (AUX/IAA; IAA). While ethylene is involved in inducible aerenchyma formation, the relationship of auxin and ethylene during aerenchyma formation remains unclear. Here, we examined the effects of oxygen deficiency and ethylene on aerenchyma formation in the roots of a rice mutant (iaa13) in which auxin signaling is suppressed by a mutation in the degradation domain of IAA13 protein. The results showed that AUX/IAA-mediated auxin signaling contributes to ethylene-dependent inducible aerenchyma formation in rice roots. An auxin transport inhibitor abolished aerenchyma formation under oxygen-deficient conditions and reduced the expression of genes encoding ethylene biosynthesis enzymes, further supporting the idea that auxin is involved in ethylene-dependent inducible aerenchyma formation. Based on these studies, we propose a mechanism that underlies the relationship between auxin and ethylene during inducible aerenchyma formation in rice roots.

Multiple Integrated Root Phenotypes Are Associated with Improved Drought Tolerance.

To test the hypothesis that multiple integrated root phenotypes would co-optimize drought tolerance, we phenotyped the root anatomy and architecture of 400 mature maize (Zea mays) genotypes under well-watered and water-stressed conditions in the field. We found substantial variation in all 23 root phenes measured. A phenotypic bulked segregant analysis revealed that bulks representing the best and worst performers in the field displayed distinct root phenotypes. In contrast to the worst bulk, the root phenotype of the best bulk under drought consisted of greater cortical aerenchyma formation, more numerous and narrower metaxylem vessels, and thicker nodal roots. Partition-against-medians clustering revealed several clusters of unique root phenotypes related to plant performance under water stress. Clusters associated with improved drought tolerance consisted of phene states that likely enable greater soil exploration by reallocating internal resources to greater root construction (increased aerenchyma content, larger cortical cells, fewer cortical cell files), restrict uptake of water to conserve soil moisture (reduced hydraulic conductance, narrow metaxylem vessels), and improve penetrability of hard, dry soils (thick roots with a larger proportion of stele, and smaller distal cortical cells). We propose that the most drought-tolerant-integrated phenotypes merit consideration as breeding ideotypes.

Tolerant mechanisms to O2 deficiency under submergence conditions in plants

Wetland plants can tolerate long-term strict hypoxia and anoxic conditions and the subsequent re-oxidative stress compared to terrestrial plants. During O2 deficiency, both wetland and terrestrial plants use NAD(P)+ and ATP that are produced during ethanol fermentation, sucrose degradation, and major amino acid metabolisms. The oxidation of NADH by non-phosphorylating pathways in the mitochondrial respiratory chain is common in both terrestrial and wetland plants. As the wetland plants enhance and combine these traits especially in their roots, they can survive under long-term hypoxic and anoxic stresses. Wetland plants show two contrasting strategies, low O2 escape and low O2 quiescence strategies (LOES and LOQS, respectively). Differences between two strategies are ascribed to the different signaling networks related to phytohormones. During O2 deficiency, LOES-type plants show several unique traits such as shoot elongation, aerenchyma formation and leaf acclimation, whereas the LOQS-type plants cease their growth and save carbohydrate reserves. Many wetland plants utilize NH4+ as the nitrogen (N) source without NH4+-dependent respiratory increase, leading to efficient respiratory O2 consumption in roots. In contrast, some wetland plants with high O2 supply system efficiently use NO3− from the soil where nitrification occurs. The differences in the N utilization strategies relate to the different systems of anaerobic ATP production, the NO2−-driven ATP production and fermentation. The different N utilization strategies are functionally related to the hypoxia or anoxia tolerance in the wetland plants.

Newly identified miRNAs may contribute to aerenchyma formation in sugarcane roots.

Small RNAs comprise three families of noncoding regulatory RNAs that control gene expression by blocking mRNA translation or leading to mRNA cleavage. Such post‐transcriptional negative regulation is relevant for both plant development and environmental adaptations. An important biotechnological application of miRNA identification is the discovery of regulators and effectors of cell wall degradation, which can improve/facilitate hydrolysis of cell wall polymers for second‐generation bioethanol production. The recent characterization of plant innate cell wall modifications occurring during root aerenchyma development triggered by ethylene led to the possibility of prospection for mechanisms of cell wall disassembly in sugarcane. By using next‐generation sequencing, 39 miRNAs were identified in root segments along the process of aerenchyma development. Among them, 31 miRNAs were unknown to the sugarcane miRBase repository but previously identified as produced by its relative Sorghum bicolor. Key putative targets related to signal transduction, carbohydrate metabolic process, and cell wall organization or biogenesis were among the most representative gene categories targeted by miRNA. They belong to the subclasses of genes associated with the four modules of cell wall modification in sugarcane roots: cell expansion, cell separation, hemicellulose, and cellulose hydrolysis. Thirteen miRNAs possibly related to ethylene perception and signaling were also identified. Our findings suggest that miRNAs may be involved in the regulation of cell wall degradation during aerenchyma formation. This work also points out to potential molecular tools for sugarcane improvement in the context of second‐generation biofuels.

High water uptake ability was associated with root aerenchyma formation in rice: Evidence from local ammonium supply under osmotic stress conditions

Root water uptake is strongly influenced by the morphology and anatomical structure of roots, which are regulated by nitrogen forms and environmental stimuli. To further illustrate the roles of different nitrogen forms on root water uptake under osmotic stress, a split-root system was supplied with different nitrogen forms and osmotic stress simulated by adding 10% (w/v) polyethylene glycol (PEG, 6000). The local effects of nitrogen form and osmotic stress on root morphology, anatomical structure, root lignin content, and water uptake rate were investigated. Under osmotic stress conditions, ammonium markedly promoted the formation and elongation of the lateral root, whereas a significant decrease in numbers of lateral roots was observed under local nitrate supply. Under nitrate supply in split-root systems, osmotic stress significantly promoted root cell death and more aerenchyma formation, as well as accelerated the lignification of the root. However, osmotic stress had no negative effect on the root anatomical structure under ammonium supply. The root water uptake rate was significantly higher in split-root supplied with ammonium than nitrate under osmotic stress conditions. In conclusion, the high water uptake ability in local ammonium supply was associated with the more lateral roots development and the lower cell death, aerenchyma formation and lignification under osmotic stress.

Sinusoidal vibration alleviates salt stress by induction of antioxidative enzymes and anatomical changes in Mentha pulegium (L.)

Effect of mechanical vibration on salt stress alleviation was studied in pennyroyal (Mentha pulegium L.) seedlings under in vitro and greenhouse conditions, and changes in growth, anatomical, and antioxidative responses were investigated. To determine the optimum frequency of vibration, the sterilized seeds were cultivated on Murashige and Skoog (MS) basal medium. The 14-day-old seedlings were exposed to various frequencies of sinusoidal vibration (0, 50, 100, and 150 Hz) for 30 min, and the optimum frequency was determined based on some growth, physiological, and anatomical parameters. Vibrations up to 100 Hz increased growth, shoot length, chlorophyll content, and relative water content (RWC). Vibration-induced aerenchyma formation, stele diameter, phloem, and suppressed xylem and only a few xylem vessels can be observed at 100 Hz. For evaluation of vibration effects on salt stress alleviation, the seedlings were treated by optimum vibration frequency (100 Hz) and then were subjected to salinity stress (0, 50, 100, and 150 mM NaCl). In salt-stressed plants, vibration decreased malondialdehyde (MDA), H2O2, electrolyte leakage, total carbohydrates, polyphenol oxidase (PPO) activity, and induced proline, protein, ascorbate peroxidase (APX) peroxidase (POX), and superoxide dismutase (SOD) activities. The lower contents of MDA, H2O2, and electrolyte leakage under in vitro grown seedlings comparing to that of whole plants could be associated with the higher levels of in vitro antioxidative enzyme activities. However, vibration induced more antioxidative enzyme activities.