Green-synthesized lignin nanoparticles enhance Zea mays resilience to salt stress by improving antioxidant metabolism and mitigating ultrastructural damage.

Soil salinity poses a substantial threat to agricultural productivity, resulting in far-reaching consequences. Green-synthesized lignin nanoparticles (LNPs) have emerged as significant biopolymers which effectively promote sustainable crop production and enhance abiotic stress tolerance. However, the defensive role and underlying mechanisms of LNPs against salt stress in Zea mays remain unexplored. The present study aims to elucidate two aspects: firstly, the synthesis of lignin nanoparticles from alkali lignin, which were characterized using Field Emission Scanning Electron Microscopy (FE-SEM), Transmission Electron Microscopy (TEM), Fourier Infrared Spectroscopy (FT-IR) and Energy Dispersive X-Ray Spectroscopy (EDX). The results confirmed the purity and morphology of LNPs. Secondly, the utilization of LNPs (200 mg/L) in nano priming to alleviate the adverse effects of NaCl (150 mM) on Zea mays seedlings. LNPs significantly reduced the accumulation of Na+ (17/21%) and MDA levels (21/28%) in shoots/roots while increased lignin absorption (30/31%), resulting in improved photosynthetic performance and plant growth. Moreover, LNPs substantially improved plant biomass, antioxidant enzymatic activities and upregulated the expression of salt-tolerant genes (ZmNHX3 (1.52 & 2.81 FC), CBL (2.83 & 3.28 FC), ZmHKT1 (2.09 & 4.87 FC) and MAPK1 (3.50 & 2.39 FC) in both shoot and root tissues. Additionally, SEM and TEM observations of plant tissues confirmed the pivotal role of LNPs in mitigating NaCl-induced stress by reducing damages to guard cells, stomata and ultra-cellular structures. Overall, our findings highlight the efficacy of LNPs as a practical and cost-effective approach to alleviate NaCl-induced stress in Zea mays plants. These results offer a sustainable agri-environmental strategy for mitigating salt toxicity and enhancing crop production in saline environments.

Sodium nitroprusside application improves morphological and physiological attributes of soybean (Glycine max L.) under salinity stress.

Salinity is among the major abiotic stresses negatively affecting the growth and productivity of crop plants. Sodium nitroprusside (SNP) -an external nitric oxide (NO) donor- has been found effective to impart salinity tolerance to plants. Soybean (Glycine max L.) is widely cultivated around the world; however, salinity stress hampers its growth and productivity. Therefore, the current study evaluated the role of SNP in improving morphological, physiological and biochemical attributes of soybean under salinity stress. Data relating to biomass, chlorophyll and malondialdehyde (MDA) contents, activities of various antioxidant enzymes, ion content and ultrastructural analysis were collected. The SNP application ameliorated the negative effects of salinity stress to significant extent by regulating antioxidant mechanism. Root and shoot length, fresh and dry weight, chlorophyll contents, activities of various antioxidant enzymes, i.e., catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) were improved with SNP application under salinity stress compared to control treatment. Similarly, plants treated with SNP observed less damage to cell organelles of roots and leaves under salinity stress. The results revealed pivotal functions of SNP in salinity tolerance of soybean, including cell wall repair, sequestration of sodium ion in the vacuole and maintenance of normal chloroplasts with no swelling of thylakoids. Minor distortions of cell membrane and large number of starch grains indicates an increase in the photosynthetic activity. Therefore, SNP can be used as a regulator to improve the salinity tolerance of soybean in salt affected soils.

Correction: Effect of cutting depth during sugarcane (Saccharum spp. hybrid) harvest on root characteristics and yield.

[This corrects the article DOI: 10.1371/journal.pone.0238085.].

Indigenous Tocopherol Improves Tolerance of Oilseed Rape to Cadmium Stress.

Two oilseed rape genotypes (Jiu-Er-13XI and Zheyou-50), differing in seed oil content, were subjected to cadmium (Cd) stress in hydroponic experiment. Genotypic differences were observed in terms of tolerance to Cd exposure. Cd treatment negatively affected both genotypes, but the effects were more devastating in Jiu-Er-13XI (low seed oil content) than in Zheyou-50 (high seed oil content). Jiu-Er-13XI accumulated more reactive oxygen species (ROS), which destroyed chloroplast structure and decreased photosynthetic pigments, than Zheyou-50. Total fatty acids, especially 18:2 and 18:3, severely decreased as suggested by increase in MDA content. Roots and shoots of Jiu-Er-13XI plants accumulated more Cd content, while less amount of tocopherol (Toc) was observed under Cd stress, than Zheyou-50. Conversely, Zheyou-50 was less affected by Cd stress than its counterpart. It accumulated comparatively less amount of Cd in roots and shoots, along with reduced accumulation of malondialdehyde (MDA) and ROS under Cd stress, than Jiu-Er-13XI. Further, the level of Toc, especially α-Tocopherol, was much higher in Zheyou-50 than in Jiu-Er-13XI, which was also supported by high expression of Toc biosynthesis genes in Zheyou-50 during early hours. Toc not only restricted the absorption of Cd by roots and its translocation to shoot but also scavenged the ROS generated during oxidative stresses. The low level of MDA shows that polyunsaturated fatty acids in chloroplast membranes remained intact. In the present study the tolerance of Zheyou-50 to Cd stress, over Jiu-Er-13XI, is attributed to the activities of Toc. This study shows that plants with high seed oil content are tolerant to Cd stress due to high production of Toc.

Characterization, Knockdown and Parental Effect of Hexokinase Gene of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) Revealed by RNA Interference.

Hexokinase (HK) is a key enzyme in chitin biosynthesis in insects and plays an important role in development and energy regulation. It also performs a crucial role in the synthesis of Glucose-6-phosphate and its putative functions are studied via injection of dsRNA corresponding to the hexokinase gene from Cnaphalocrocis medinalis (CmHK). This study was designed to analyze the characteristics and expression patterns of HK-related genes in various tissues of C. medinalis at different developmental stages. The CmHK ORF is a 1359 bp in length, encoding a protein of 452 amino acids, with homology and cluster analysis showing that CmHK shares an 85.11% sequence similarity with hexokinase from Ostrinia furnacalis.CmHK was highly expressed in the ovary and in the fifth instar larvae. Injection of dsCmHK significantly suppressed mRNA expression (73.6%) 120 h post-dsRNA injection as compared to a control group. The results demonstrated an increased incidence of larval and pupal mortality of 80% and 78%, respectively, with significant variation in the sex ratio between males (68.33%) and females (35%), overt larval deformities, and a reduction in average weight gain observed 120 h post-dsRNA injection. In addition, dsCmHK-injected C. medinalis showed a significant reduction in ovulation per female and larval hatching rate, along with increased larval and pupal mortality and variation in male and female emergence over three generations (G1, G2, and G3). Taken together, the outcomes of the study provide a foundation to study gene function and a new dimension to control C. medinalis by transgenic RNAi technology.

Calmodulin-binding transcription activator (CAMTA) genes family: Genome-wide survey and phylogenetic analysis in flax (Linum usitatissimum).

Flax (Linum usitatissimum) is a member of family linaceae with annual growth habit. It is included among those crops which were domesticated very early and has been used in development related studies as a model plant. In plants, Calmodulin-binding transcription activators (CAMTAs) comprise a unique set of Calmodulin-binding proteins. To elucidate the transport mechanism of secondary metabolites in flax, a genome-based study on these transporters was performed. The current investigation identified nine CAMTAs proteins, classified into three categories during phylogenetic analysis. Each group had significant evolutionary role as illustrated by the conservation of gene structures, protein domains and motif organizations over the distinctive phylogenetic classes. GO annotation suggested a link to sequence-specific DNA and protein binding, response to low temperature and transcription regulation by RNA polymerase II. The existence of different hormonal and stress responsive cis-regulatory elements in promotor region may directly correlate with the variation of their transcripts. MicroRNA target analysis revealed that various groups of miRNA families targeted the LuCAMTAs genes. Identification of CAMTA genes, miRNA studies and phylogenetic analysis may open avenues to uncover the underlying functional mechanism of this important family of genes in flax.

Drought-induced alterations in photosynthetic, ultrastructural and biochemical traits of contrasting sugarcane genotypes.

Drought is an important factor which limits growth of sugarcane. To elucidate the physiological and biochemical mechanisms of tolerance, a pot experiment was conducted at Sugarcane Research Institute, Kaiyuan, China. Two genotypes (Yuetang 93-159-sensitive and Yunzhe 05-51-tolerant), were subjected to three treatments; 70±5% (control), 50±5% (moderate drought) and 30±5% (severe drought) of soil field capacity. The results demonstrated that drought induced considerable decline in morpho-physiological, biochemical and anatomical parameters of both genotypes, with more pronounced detrimental effects on Yuetang 93-159 than on Yunzhe 05-51. Yunzhe 05-51 exhibited more tolerance by showing higher dry biomass, photosynthesis and antioxidant enzyme activities. Compared with Yuetang 93-159, Yunzhe 05-51 exhibited higher soluble sugar, soluble protein and proline contents under stress. Yunzhe 05-51 illustrated comparatively well-composed chloroplast structure under drought stress. It is concluded that the tolerance of Yunzhe 05-51 was attributed to improved antioxidant activities, osmolyte accumulation and enhanced photosynthesis. These findings may provide valuable information for future studies on molecular mechanism of tolerance.

Comparative study of the genetic basis of nitrogen use efficiency in wild and cultivated barley.

To curb the increasing demand for nitrogenous fertilizers, it is imperative to develop new cultivars with comparatively greater nitrogen use efficiency (NUE). Nonetheless, so far very meager information is available concerning the variances among barley (Hordeum vulgare L.) varieties for their response to nitrogen deprivation. The current study was carried out to explore the potential of barley genotypes for higher NUE. A hydroponic experiment was conducted at seedling stage to compare the performance of four barley genotypes, ZD9 and XZ149 (with higher NUE) and HXRL and XZ56 (with lower NUE) in response to low (0.1 mM) and normal nitrogen (2 mM) levels. Under low N, all the genotypes expressed less number of tillers, decreased soluble proteins, chlorophyll and N concentrations in both roots and shoots, in comparison with normal N supply. However, significant differences were found among the genotypes. The genotypes with high NUE (ZD9 and XZ149) showed higher N concentration, increased number of tillers, improved chlorophyll and soluble proteins in both roots and shoots as compared to the inefficient ones (HXRL and XZ56). Furthermore, nitrate transporter gene (NRT2.1) showed higher expression under low N, both in roots and leaves of N efficient genotypes, as compared to the N inefficient ones. However, N assimilatory genes (GS1 and GS2) showed higher expression under normal and low N level, in leaves and roots respectively. The outcome of the study revealed that genotypes with higher NUE (ZD9 and XZ149) performed better under reduced N supply, and may require relatively less N fertilizer for normal growth and development, as compared to those with lower NUE. The study also revealed a time-specific expression pattern of studied genes, indicating the duration of low N stress. The current study suggested that future work must involve the time course as a key factor while studying expression patterns of these genes to better understand the genetic basis of low-N tolerance.

Zinc-lysine prevents chromium-induced morphological, photosynthetic, and oxidative alterations in spinach irrigated with tannery wastewater.

Anthropogenic activities have resulted in severe environmental degradation. Untreated wastewater from tanneries is hazardous to all kinds of life on earth. Effluent from tanning industries, containing large amount of Cr, is used to irrigate the crops in Pakistan. The current experiment was carried out to study the effects of tannery wastewater on spinach and the role of lysine-Zn in mitigating the severity of stress. The plants were grown in soil and the following treatments were used: irrigation with 0%, 33%, 66%, and 100% wastewater (ww) along with two doses (0 mM, 10 mM) of Zn-lysine. Foliar application of zinc-lysine enhanced the plant growth, biomass, Zn contents, photosynthesis, and enzyme activities in different tissues of plant. Zinc-lysine (10 mM) considerably decreased the Cr content in roots and shoots, along with ameliorating the oxidative stress by enhancing the activities of antioxidant enzymes in plants. Addition of Zn-lys (10 mM) improved the plant height by 19%, root length by 57%, leaf dry weight by 19%, and root dry weight by33% under 100% Cr treatment. Zn-lys significantly reduces the oxidative stress and concentration of Cr as compared with the Cr treatments alone. Application of Zn-lys (10 mM) reduced the Cr contents in roots by 27 and 22 under 33 and 66% Cr treatment, respectively. Taken together, Zn-lys chelates efficiently ameliorated the toxic effects of chromium. Zn-lysine has the extravagant potential of mitigating the heavy metal toxicity without harming the normal growth and development of the plants.

Early maturing Bt cotton requires more potassium fertilizer under water deficiency to augment seed-cotton yield but not lint quality.

Exhaustive crops such as cotton require potassium (K) in copious amounts as compared to other crops. High yielding cultivars in cotton-wheat cropping system, have further increased its demand in cotton growing areas of Pakistan. As cotton is grown in arid and semiarid areas, therefore often prone to water deficiency. The reproductive growth particularly flowering and boll setting are highly sensitive to low soil water potentials, where enough K supply can play a vital role. In this two-year field studies, three cultivars (early, mid and late maturing) were cultivated at two K fertilizer levels 100, 200 kg K ha-1 along with control with no K fertilizer application at two irrigation levels. In first irrigation level, water was applied as per full irrigation schedule, while in water deficit irrigation water was applied at deficit irrigation schedule started after flowering till harvesting. It has been revealed that K application has impact on boll setting as well as seed cotton yield, however early and mid-maturing cultivars are more responsive to K fertilization. Furthermore, irrigation level had significant impact against K fertilization and relatively better response was observed in deficit irrigation as compared to full irrigation. Nevertheless, fiber quality parameters were unaffected by K fertilization. Considering the best benefit cost ratio under water deficiency, it is concluded that 100 kg K2O ha-1 should be applied at the time of seed bed preparation for economical seed-cotton yield of early maturing Bt cotton.

Combined application of citric acid and 5-aminolevulinic acid improved biomass, photosynthesis and gas exchange attributes of sunflower (Helianthus annuus L.) grown on chromium contaminated soil.

Phytoremediation is an important technique to remove heavy metals from contaminated soils due to its efficiency and cost-effectiveness. The present study was conducted to assess the synergistic role of 5-aminolevulinic acid (ALA) and citric acid (CA) in improving the phyto-extraction of chromium (Cr) by sunflower. Sunflower plants were grown in soil, spiked with different concentrations of Cr (0, 5, 10, 20 mg kg-1). Various concentrations of 5-ALA (0, 10, 20 mg L-1) and CA (0, 2.5, 5 mM) were applied exogenously at juvenile stage. A significant decrease was observed in biomass and agronomic traits of sunflower under Cr stress alone. Further, Cr toxicity significantly decreased the plant growth, soluble proteins and photosynthetic pigments. However, exogenously applied ALA and CA significantly improved the plants' physiological as well as agronomic attributes by lowering the production of reactive oxygen species and reducing electrolyte leakage. Moreover, Cr uptake was increased with increasing concentration of Cr in spiked soil, which was further enhanced by combined application of ALA and CA.

Phyto-management of chromium contaminated soils through sunflower under exogenously applied 5-aminolevulinic acid.

Soil contamination with heavy metals is threatening the food security around the globe. Chromium (Cr) contamination results in poor quality and reduction in yield of crops. The present research was performed to figure out the Cr toxicity in sunflower and the ameliorative role of 5-aminolevulinic acid (ALA) as a plant growth regulator. The sunflower (FH-614) was grown under increasing concentration of Cr (0, 5, 10 and 20mgkg-1) alone and/or in combination with 5-ALA (0, 10 and 20mgL-1). Results showed that Cr suppressed the overall growth, biomass, gas exchange attributes and chlorophyll content of sunflower plants. Moreover, lower levels of Cr (5 and 10mgkg-1) increased the production of reactive oxygen species (ROS) and electrolyte leakage (EL) along with the activities of antioxidant enzymes i.e., superoxide dismutase (SOD), guaiacole peroxidase (POD), ascorbate (APX), catalase (CAT). But at higher concentration of Cr (20mgkg-1), the activities of these enzymes presented a declining trend. However, the addition of 5-ALA significantly alleviated the Cr-induced toxicity in sunflower plant and enhanced the plant growth and biomass parameters along with increased chlorophyll content, gas exchange attributes, soluble proteins and soil plant analysis development (SPAD) values by scavenging the ROS and lowering down the EL. The 5-ALA also enhanced the activities of antioxidant enzymes at all levels of Cr. The increase in Cr concentration in all plant parts such as leaf, root and stem was directly proportional to the Cr concentration in soil. The application of 5-ALA further enhanced the uptake of Cr and its concentration in the plants. To understand this variation in response of plants to 5-ALA, detailed studies are required on plant biochemistry and genetic modifications.

Citric acid assisted phytoextraction of chromium by sunflower; morpho-physiological and biochemical alterations in plants.

Soil and water contamination from heavy metals and metalloids is one of the most discussed and burning global issues due to its potential to cause the scarcity of healthy food and safe water. The scientific community is proposing a range of lab and field based physical, chemical and biological solutions to remedy metals and metalloids contaminated soils and water. The present study finds out a possibility of Chromium (Cr) extraction by sunflower from spiked soil under chelating role of citric acid (CA). The sunflower plants were grown under different concentrations of Cr (0, 5, 10 & 20mgkg-1) and CA (0, 2.5 & 5mM). Growth, biomass, gas exchange, photosynthesis, electrolyte leakage (EL), reactive oxygen species (ROS; malondialdehyde (MDA), hydrogen peroxide (H2O2) and the activities of antioxidant enzymes such as, superoxide dismutase (SOD), guaiacole values peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT) were measured. The results depicted a clear decline in plant height, root length, leaf area, number of leaves and flowers per plant along with fresh and dry biomass of all parts of plant with increasing concentration of Cr in soil. Similar reduction was observed in chlorophyll a and b, total chlorophyll, carotenoids, soluble protein, gas exchange attributes and SPAD. The increasing concentration of Cr also enhanced the Cr uptake and accumulation in plant roots, stem and leaves along with the production of ROS and EL. The activities of antioxidant enzymes increased with increasing Cr concentration from 0 to 10mg, but decreased at 20mgkg-1 soil. The CA application significantly alleviated Cr-induced inhibition of plant growth, biomass, photosynthesis, gas exchange, soluble proteins and SPAD value. Presence of CA also enhanced the activities of all antioxidant enzymes and reduced the production of ROS and EL. The chelating potential of CA increased the concentration and accumulation of Cr in plant roots, stem and leaves. It is concluded that the sunflower can be a potential candidate for the remediation of Cr under CA treatment, while the possibility may vary with genotype, Cr level and CA concentration.

Phyto-management of Cr-contaminated soils by sunflower hybrids: physiological and biochemical response and metal extractability under Cr stress.

Chromium (Cr) is a biologically non-essential, carcinogenic and toxic heavy metal. The cultivation of Cr-tolerant genotypes seems the most favorable and environment friendly strategy for rehabilitation and remediation of Cr-contaminated soils. To prove this hypothesis and identify the Cr tolerance, the present study was performed to assess the physiological and biochemical response of sunflower genotypes to Cr stress. The seeds of six sunflower hybrids, namely FH-425, FH-600, FH-612, FH-614, FH-619, and FH-620, were grown in spiked soil for 12 weeks under increasing concentrations of Cr (0, 5, 10, and 20 mg kg-1). A seed germination test was also run under different concentrations of Cr (0, 5, 10, 200 mM) in petri dishes. Plants were harvested after 12 weeks of germination. Different plant attributes such as growth; biomass; photosynthesis; gas exchange; activity of antioxidant enzymes, i.e., superoxide dismutase (SOD), guaiacol peroxidase (POD), ascorbate (APX), and catalases (CAT); reactive oxygen species (ROS); lipid peroxidation; electrolyte leakage; and Cr concentration as well as accumulations in all plant parts were studied for the selection of the most Cr-tolerant genotype. Increasing concentration of Cr in soil triggered the reduction of all plant parameters in sunflower. Cr stress increased electrolyte leakage and production of reactive oxygen species which stimulated the activities of antioxidant enzymes and gas exchange attributes of sunflower. Chromium accumulation in the root and shoot increased gradually with increasing Cr treatments and caused reduction in overall plant growth. The accumulation of Cr was recorded in the order of FH-614 > FH-620 > FH-600 > FH-619 > FH-612 > FH-425. The differential uptake and accumulation of Cr by sunflower hybrids may be useful in selection and breeding for Cr-tolerant genotypes.

Citric acid enhanced the antioxidant defense system and chromium uptake by Lemna minor L. grown in hydroponics under Cr stress.

Phytoextraction is a cost-effective and eco-friendly technique for the removal of pollutants, mainly heavy metal(loids) especially from polluted water and metal-contaminated soils. The phytoextraction of heavy metals is, in general, limited due to the low availability of heavy metals in the growth medium. Organic chelators can help to improve the phytoextraction by increasing metal mobility and solubility in the growth medium. The present research was carried out to examine the possibility of citric acid (CA) in improving chromium (Cr) phytoextraction by Lemna minor (duckweed). For this purpose, healthy plants were collected from nearby marsh and grown in hydroponics under controlled conditions. Initial metal contents of both marsh water and plant were measured along with physico-chemical properties of the marsh water. Different concentrations of Cr and CA were applied in the hydroponics in different combinations after defined intervals. Continuous aeration was supplied and pH maintained at 6.5 ± 0.1. Results showed that increasing concentration of Cr significantly decreased the plant biomass, photosynthetic pigments, leaf area, and antioxidant enzyme activities (like catalase, ascorbate peroxidase, superoxide dismutase, peroxidase). Furthermore, Cr stress increased the Cr concentrations, electrolyte leakage, hydrogen peroxide, and malondialdehyde contents in plants. The addition of CA alleviated the Cr-induced toxicity in plants and further enhanced the Cr uptake and its accumulation in L. minor. The addition of CA enhanced the Cr concentration in L. minor by 6.10, 26.5, 20.5, and 20.2% at 0, 10, 100, and 200 µM Cr treatments, respectively, compared to the respective Cr treatments without CA. Overall, the results of the present study showed that CA addition may enhance the Cr accumulation and tolerance in L. minor by enhancing the plant growth and activities of antioxidant enzymes.

Genotype-dependent effect of exogenous 24-epibrassinolide on chromium-induced changes in ultrastructure and physicochemical traits in tobacco seedlings.

Greenhouse hydroponic experiments were carried out using three different heavy metal accumulation tobacco genotypes to evaluate how different genotypes responded to chromium (Cr) toxicity in the presence of 24-epibrassinolide (EBR; a biologically active brassinosteroid). The results showed that Cr stress caused a marked reduction in plant biomass, chlorophyll content, chlorophyll fluorescence, and photosynthesis parameters but induced malondialdehyde accumulation and ultrastructure damage, with 2010-38 (L) less affected. Foliar application of 24-epibrassinolide (0.1 µM) on Cr-stressed plants greatly alleviated Cr-induced inhibition of growth and photosynthesis, oxidative stress and ultrastructure damage, decreased Cr accumulation in different parts of leaves and roots, with the exception of the upper and lower of leaves of genotype L, and maintained ion homeostasis. Regarding genotypes, L was more tolerant than M and H, as it absorbed less Cr and also performed better in all of the studied parameters. These findings suggest a potential role for 24-epibrassinolide in Cr stress alleviation and the utilization of elite genetic resources in future breeding programs to develop low Cr accumulation genotypes. These results advocate a positive role for 24-epibrassinolide in reducing pollutant residues from health point of view.

Genome-wide identification of chromium stress-responsive micro RNAs and their target genes in tobacco (Nicotiana tabacum) roots.

Tobacco easily accumulates certain heavy metals in leaves and thus poses a potential threat to human health. To systematically dissect Cr-responsive microRNAs (miRNAs) and their targets at the global level, 4 small RNA libraries were constructed from the roots of Cr-treated (Cr) and Cr-free (control) for 2 contrasting tobacco genotypes,Yunyan2 (Cr-sensitive) and Guiyan1 (Cr-tolerant). Using high-throughput-sequencing-technology, the authors identified 53 conserved and 29 novel miRNA families. Comparative genomic analysis of 41 conserved Cr-responsive miRNA families revealed that 11 miRNA families showed up-regulation in Guiyan1 but unaltered in Yunyan2, and 17 miRNA families were up-regulated only in Yunyan2 under Cr stress. Only 1 family, miR6149, was down-regulated in Yunyan2 but remained unchanged in Guiyan1. Of the 29 novel miRNA families, 14 expressed differently in the 2 genotypes under Cr stress. Based on a high-throughput degradome sequencing homology search, potential targets were predicted for the 41 conserved and 14 novel Cr-responsive miRNA families. Clusters of Orthologous Groups functional category analysis revealed that some of these predicted target transcripts of miRNAs are responsive to biotic and abiotic stresses. Furthermore, the expression patterns of many Cr-responsive miRNAs were validated by stem-loop real-time transcription polymerase chain reaction. The results of the present study provide valuable information and a framework for understanding the function of miRNAs in Cr tolerance.