Implanting toric implantable collamer lens displays better astigmatism correction than implantable collamer lens combined with manually limbal relaxing incision.

BACKGROUND: This retrospective study aimed to compare the outcomes of toric implantable collamer lens (TICL) surgery with those of implantable collamer lens (ICL) implantation combined with limbal relaxing incision (LRI) in patients with low myopia and astigmatism. METHODS: A total of 40 eyes of 28 patients who underwent TICL implantation and 40 eyes of 27 patients who underwent ICL implantation combined with manually LRI between 2021 and 2022 were included. Primary outcomes were manifest sphere and cylinder, intraocular pressure, visual acuity, and astigmatism parameters at 1 day, 1 week, and 1, 3, and 6 months postoperatively. RESULTS: The two surgeries showed comparable effects on manifest sphere and cylinder, intraocular pressure, and visual acuity (all p > 0.1). Surgery-induced astigmatism (SIA) was maintained as stable in the TICL group (1.73 to 1.68, p = 0.420), but was significantly reduced in the ICL/LRI group (1.74 to 1.17, p = 0.001) from preoperative to postoperative 6 months. The TICL group displayed significantly higher SIA and correction index at postoperative 1, 3, and 6 months than the ICL/LRI group (at 6 months: SIA, 1.68 (1.26, 1.96) vs., 1.17 (1.00, 1.64), p = 0.010; CI: 0.98 (0.78, 1.25) vs. 0.80 (0.61, 1.04), p = 0.018). No complications occurred during follow-up. CONCLUSIONS: The effects of ICL/LRI are comparable to those of TICL in correcting myopia. TICL implantation displays better astigmatism correction than ICL/LRI.

Reference genomes of channel catfish and blue catfish reveal multiple pericentric chromosome inversions.

BACKGROUND: Channel catfish and blue catfish are the most important aquacultured species in the USA. The species do not readily intermate naturally but F1 hybrids can be produced through artificial spawning. F1 hybrids produced by mating channel catfish female with blue catfish male exhibit heterosis and provide an ideal system to study reproductive isolation and hybrid vigor. The purpose of the study was to generate high-quality chromosome level reference genome sequences and to determine their genomic similarities and differences. RESULTS: We present high-quality reference genome sequences for both channel catfish and blue catfish, containing only 67 and 139 total gaps, respectively. We also report three pericentric chromosome inversions between the two genomes, as evidenced by long reads across the inversion junctions from distinct individuals, genetic linkage mapping, and PCR amplicons across the inversion junctions. Recombination rates within the inversional segments, detected as double crossovers, are extremely low among backcross progenies (progenies of channel catfish female × F1 hybrid male), suggesting that the pericentric inversions interrupt postzygotic recombination or survival of recombinants. Identification of channel catfish- and blue catfish-specific genes, along with expansions of immunoglobulin genes and centromeric Xba elements, provides insights into genomic hallmarks of these species. CONCLUSIONS: We generated high-quality reference genome sequences for both blue catfish and channel catfish and identified major chromosomal inversions on chromosomes 6, 11, and 24. These perimetric inversions were validated by additional sequencing analysis, genetic linkage mapping, and PCR analysis across the inversion junctions. The reference genome sequences, as well as the contrasted chromosomal architecture should provide guidance for the interspecific breeding programs.

Genomic imprinting-like monoallelic paternal expression determines sex of channel catfish.

The X and Y chromosomes of channel catfish have the same gene contents. Here, we report allelic hypermethylation of the X chromosome within the sex determination region (SDR). Accordingly, the X-borne hydin-1 gene was silenced, whereas the Y-borne hydin-1 gene was expressed, making monoallelic expression of hydin-1 responsible for sex determination, much like genomic imprinting. Treatment with a methylation inhibitor, 5-aza-dC, erased the epigenetic marks within the SDR and caused sex reversal of genetic females into phenotypic males. After the treatment, hydin-1 and six other genes related to cell cycle control and proliferative growth were up-regulated, while three genes related to female sex differentiation were down-regulated in genetic females, providing additional support for epigenetic sex determination in catfish. This mechanism of sex determination provides insights into the plasticity of genetic sex determination in lower vertebrates and its connection with temperature sex determination where DNA methylation is broadly involved.

Genetic and epigenetic regulation of growth, reproduction, disease resistance and stress responses in aquaculture.

Major progress has been made with genomic and genetic studies in aquaculture in the last decade. However, research on epigenetic regulation of aquaculture traits is still at an early stage. It is apparent that most, if not all, aquaculture traits are regulated at both genetic and epigenetic levels. This paper reviews recent progress in understanding of genetic and epigenetic regulation of important aquaculture traits such as growth, reproduction, disease resistance, and stress responses. Although it is challenging to make generalized statements, DNA methylation is mostly correlated with down-regulation of gene expression, especially when at promoters and enhancers. As such, methylation of growth factors and their receptors is negatively correlated with growth; hypomethylation of genes important for stress tolerance is correlated with increased stress tolerance; hypomethylation of genes important for male or female sex differentiation leads to sex differentiation into males or females, respectively. It is apparent that environmental regulation of aquaculture traits is mediated at the level of epigenetic regulation, and such environment-induced epigenetic changes appeared to be intergenerationally inherited, but evidences for transgenerational inheritance are still limited.

Comparison of pre-chop technique using a reverse chopper and classic stop-and-chop technique in the treatment of high myopia associated with nuclear cataract.

BACKGROUND: To evaluate the safety and efficacy of the pre-chop technique using a novel reverse chopper vs. the classic stop-and-chop technique in phacoemulsification for patients with high myopia and associated grade III-IV nuclear cataracts. METHODS: In this prospective cohort study, a total of 44 consecutive patients (44 eyes) with grade III-IV nuclear cataracts who were admitted to our hospital for cataract surgery between March 2018 and September 2018 were enrolled. All patients had ocular axial length > 27 mm and myopic refraction more than -10 diopters. Patients were randomly divided into a pre-chop group and stop-and-chop group using a randomization table. Nucleus splitting was performed surgically in both groups using either the pre-chop technique with reverse chopper or the classic stop-and-chop technique. RESULTS: Postoperative visual acuity was significantly improved in both groups compared with preoperative values. Significantly better visual acuity, lower degree of corneal edema and lower rates of corneal endothelial cell loss were observed in the pre-chop group compared to those in the classic stop-and-chop group. No complications were reported in either group. CONCLUSIONS: In treating patients with high myopia associated with grade III-IV cataracts, the pre-chop technique using a reverse chopper reduces damage to corneal endothelial cells and improves visual acuity better than the classic stop-and-chop technique.

Feminization of channel catfish with 17ß-oestradiol involves methylation and expression of a specific set of genes independent of the sex determination region.

Exogenous oestrogen 17ß-oestradiol (E2) has been shown to effectively induce feminization in teleosts. However, the molecular mechanisms underlying the process remain unclear. Here, we determined global DNA methylation and gene expression profiles of channel catfish (Ictalurus punctatus) during early sex differentiation after E2 treatment. Overall, the levels of global DNA methylation after E2 treatment were not significantly different from those of controls. However, a specific set of genes were differentially methylated, which included many sex differentiation-related pathways, such as MARK signalling, adrenergic signalling, Wnt signalling, GnRH signalling, ErbB signalling, and ECM-receptor interactions. Many genes involved in these pathways were also differentially expressed after E2 treatment. Specifically, E2 treatments resulted in upregulation of female-related genes and downregulation of male-related genes in genetic males during sex reversal. However, E2-induced sex reversal did not cause sex-specific changes in methylation profiles or gene expression within the sex determination region (SDR) on chromosome 4, suggesting that E2-induced sex reversal was a downstream process independent of the sex determination process that was regulated by sex-specific methylation within the SDR.

Identification of an Epigenetically Marked Locus within the Sex Determination Region of Channel Catfish.

Channel catfish has an XY sex determination system. However, the X and Y chromosomes harbor an identical gene content of 950 genes each. In this study, we conducted comparative analyses of methylome and transcriptome of genetic males and genetic females before gonadal differentiation to provide insights into the mechanisms of sex determination. Differentially methylated CpG sites (DMCs) were predominantly identified on the sex chromosome, most notably within the sex determination region (SDR), although the overall methylation profiles across the entire genome were similar between genetic males and females. The drastic differences in methylation were located within the SDR at nucleotide position 14.0-20.3 Mb of the sex chromosome, making this region an epigenetically marked locus within the sex determination region. Most of the differentially methylated CpG sites were hypermethylated in females and hypomethylated in males, suggesting potential involvement of methylation modification in sex determination in channel catfish. Along with the differential methylation in the SDR, a number of differentially expressed genes within the SDR were also identified between genetic males and females, making them potential candidate genes for sex determination and differentiation in channel catfish.

Allelically and Differentially Expressed Genes After Infection of Edwardsiella ictaluri in Channel Catfish as Determined by Bulk Segregant RNA-Seq.

Identification of genetic markers associated with resistance against enteric septicemia of catfish (ESC) is of great interest for genetic enhancement programs of catfish. In the present study, bulk segregant RNA-Seq analysis was applied to determine differentially expressed genes and alleles after ESC infection. Here we report three genomic regions on LG1, LG12, and LG26, containing significant single-nucleotide polymorphisms (SNPs). These genomic regions aligned well with quantitative trait loci (QTL) previously identified. Within the QTL regions, eleven genes were found to be differentially regulated between phenotypic bulks. Importantly, the QTL on linkage group 1 (LG1) were found to be expressed in the liver, whereas the QTL on LG12 and LG26 were expressed in the intestine, suggesting multiple mechanisms of ESC resistance. It is apparent that apolipoproteins may be important for ESC resistance as the QTL on LG1 included the 14-kDa apolipoprotein genes that are both allelically expressed and differentially expressed between the resistant and susceptible bulks. Traf2 and NCK-interacting protein kinase (TNIK) were found in the QTL on LG12, and it was downregulated in resistant fish, suggesting the importance of NCK downregulation in ESC resistance, as previously reported. In addition, we observed divergent gene expression patterns between the liver and intestine after infection. Immune/inflammatory-related processes were overrepresented from liver DEGs, while those DEGs identified from intestine were enriched for proteolysis and wounding processes. Taken together, the BSR-Seq analysis presented here advanced the knowledge of ESC resistance, providing information of not only positions of QTL but also genes and their differential expression between resistant and susceptible fish, making it one step closer to the identification of the causal genes for ESC resistance.

Crosstalk between dopamine and insulin signaling in growth control of the oyster.

Neuroendocrine hormones such as dopamine and insulin/insulin-like peptides play indispensable roles in growth regulation of animals, while the interplay between dopamine and insulin signaling pathways remains largely unknown in invertebrates. In the present study, we showed that tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis, was highly expressed in all tissues of the fast-growing oysters, and gradually increased with the development, which indicated the potential role of dopamine in growth regulation. Incubated with dopamine hydrochloride and insulin-like peptide recombinant proteins in vitro induced the expression of TH, suggesting a mutual regulatory relationship between insulin and dopamine signaling. Fasting and re-feeding experiments confirmed the role of TH in food intake regulation, also provide a clue about the potential regulatory relationship between the FoxO and TH. Further luciferase assay experiment confirmed that FoxO was involved in transcriptional regulation of TH gene through binding to its specific promoter region. This work provided insights into the crosstalk between dopamine and insulin signaling in growth control of mollusks.

Application of a prechop technique using a reverse chopper in small pupil cataract surgery.

BACKGROUND: To analyze the value of a prechop technique for splitting the nucleus of the lens using a reverse chopper in small-pupil cataract surgeries. A prospective case-control study. Thirty-four cataract patients (34 eyes) who were treated in our center from March 2019 to December 2019 were enrolled and then divided into two groups: small pupil group (18 patients; 18 eyes) and normal pupil group (16 patients; 16 eyes). METHODS: The prechop technique was applied in both groups, and the patients were followed up for three months. The best-corrected visual acuity (BVCA), surgical complications, corneal endothelial cell loss, pupil function, operative time, and cumulative dissipated energy (CDE) were compared between these two groups, and the safety of the nucleus-chopping technique was evaluated. Data were analyzed using the SPSS 23.0 software packages. BVCA, surgical complications, rate of corneal endothelial cell loss, pupil function, operative time, and CDE. RESULTS: The surgery was smooth in all cases. The operative time, intraoperative ultrasound energy consumption, BVCA, and surgical complications indicated no significant difference between the two groups (all P>0.05). In the small pupil group, BVCA was significantly improved after surgery and achieved its optimal value three months after surgery (χ2=49.38; P=0). The diameter of the pupil was about 3.22 mm in the small pupil group before nucleus chopping. The postoperative pupil morphology was not statistically different from that before surgery (pupil morphology: χ2=0.131; P=0.717); however, the pupillary light reflex was significantly improved after surgery (χ2=8.378; P=0.004), and the pupil diameter was significantly increased (T=-3.494; P=0.003). The rate of corneal endothelial cell loss was higher in small pupil group than in the normal pupil group in the 3rd postoperative month, but the difference was not statistically significant (T=-0.023; P=0.982). CONCLUSIONS: The prechop technique using a reverse chopper in small-pupil cataract surgery occupies a similar operative time, cumulative energy consumption during operation, BVCA, pupil morphology, and rate of corneal endothelial cell loss, comparing with those in normal-pupil cataract surgery. To such a degree, it is a safe, high-efficiency, a simple and easy-to-operate nucleus-chopping technique that can be used in small-pupil cataract surgery.

Site-selective electrooxidation of methylarenes to aromatic acetals.

Aldehyde is one of most synthetically versatile functional groups and can participate in numerous chemical transformations. While a variety of simple aromatic aldehydes are commercially available, those with a more complex substitution pattern are often difficult to obtain. Benzylic oxygenation of methylarenes is a highly attractive method for aldehyde synthesis as the starting materials are easy to obtain and handle. However, regioselective oxidation of functionalized methylarenes, especially those that contain heterocyclic moieties, to aromatic aldehydes remains a significant challenge. Here we show an efficient electrochemical method that achieves site-selective electrooxidation of methyl benzoheterocycles to aromatic acetals without using chemical oxidants or transition-metal catalysts. The acetals can be converted to the corresponding aldehydes through hydrolysis in one-pot or in a separate step. The synthetic utility of our method is highlighted by its application to the efficient preparation of the antihypertensive drug telmisartan.

Genomic Prediction of Columnaris Disease Resistance in Catfish.

Catfish is an important aquaculture species in the USA. Columnaris disease is distributed worldwide, affecting a wide variety of fish species including catfish . It leads to huge economic losses each year to the US catfish industry. Channel catfish in general is highly resistant to the disease, while blue catfish is highly susceptible. Genomic selection is an effective and accurate way to predict the breeding values and thus was expected to improve the prediction veracity of columnaris disease resistance in catfish effectively. In this study, two different methods, elastic net genomic best linear unbiased prediction (ENGBLUP) and genomic best linear unbiased prediction (GBLUP), were used to predict the columnaris disease resistance evaluated by binary survival status. Cross-validation showed that the prediction accuracy of ENGBLUP and GBLUP was 0.7347 and 0.4868, respectively, showing that ENGBLUP had a high prediction accuracy. It was shown that fitting QTL and polygenic effect with different distribution will improve genomic prediction accuracy for binary traits. In this study, an accurate and effective genomic selection method was proposed to predict the columnaris resistance in catfish, and its application should be beneficial to catfish breeding.

Post-transcriptional regulation through alternative splicing after infection with Flavobacterium columnare in channel catfish (Ictalurus punctatus).

Columnaris disease has long been recognized as a serious problem worldwide which affects both wild and cultured freshwater fish including the commercially important channel catfish (Ictalurus punctatus). The fundamental molecular mechanisms of the host immune response to the causative agent Flavobacterium columnare remain unclear, though gene expression analysis after the bacterial infection has been conducted. Alternative splicing, a post-transcriptional regulation process to modulate gene expression and increase the proteomic diversity, has not yet been studied in channel catfish following infection with F. columnare. In this study, genomic information and RNA-Seq datasets of channel catfish were used to characterize the changes of alternative splicing after the infection. Alternative splicing was shown to be induced by F. columnare infection, with 8.0% increase in alternative splicing event at early infection stage. Intriguingly, genes involved in RNA binding and RNA splicing themselves were significantly enriched in differentially alternatively spliced (DAS) gene sets after infection. This finding was consistent with our previous study in channel catfish following infection with Edwardsiella ictaluri. It was suggested to be a universal mechanism that genes involved in RNA binding and splicing were regulated to undergo differential alternative splicing after stresses in channel catfish. Moreover, many immune genes were observed to be differentially alternatively spliced after infection. Further studies need to be performed to get a deeper view of molecular regulation on alternative splicing after stresses, setting a foundation for developing catfish broodstocks with enhanced disease resistance.

Polyadenylation sites and their characteristics in the genome of channel catfish (Ictalurus punctatus) as revealed by using RNA-Seq data.

Polyadenylation plays important roles in gene expression regulation in eukaryotes, which typically involves cleavage and poly(A) tail addition at the polyadenylation site (PAS) of the pre-mature mRNA. Many eukaryotic genes contain more than one PASs, termed as alternative polyadenylation (APA). As a crucial post-transcriptional regulation, polyadenylation affects various aspects of RNA metabolism such as mRNA stability, translocation, and translation. However, polyadenylation has been rarely studied in teleosts. Here we conducted polyadenylation analysis in channel catfish, a commercially important aquaculture species around the world. Using RNA-Seq data, we identified 20,320 PASs which were classified into 14,500 clusters by merging adjacent PASs. Most of the PASs were found in 3' UTRs, followed by intron regions based on the annotation of channel catfish reference genome. No apparent difference in PAS distribution was observed between the sense and antisense strand of the channel catfish genome. The sequence analysis of nucleotide composition and motif around PASs yielded a highly similar profile among various organisms, suggesting the conservation and importance of polyadenylation in evolution. Using APA genes with more than two PASs, gene ontology enrichment revealed genes particularly involved in RNA binding. Reactome pathway analysis showed the enrichment of the innate immune system, especially neutrophil degranulation.

Biomimetic Enantioselective Total Synthesis of (-)-Robustanoids A and B and Analogues.

We report a step-economical, enantioselective total synthesis of (-)-robustanoid B and (-)-robustanoid A and four novel natural product-like compounds. Our strategy relied on our biosynthetic hypothesis and on a novel complexity generation methodology, namely, the one-pot hydroxylative double cyclization reaction. The latter consists of a modified 3,3-dimethyldioxirane-triggered epoxidation-epoxide-ring-opening cyclization reaction cascade and Trost's regioselectivity umpolung methodology ("anti-Michael addition").

GWAS Analysis Indicated Importance of NF-κB Signaling Pathway in Host Resistance Against Motile Aeromonas Septicemia Disease in Catfish.

Motile Aeromonas septicemia (MAS) disease caused by a bacterial pathogen, Aeromonas hydrophila, is an emerging but severe disease of catfish. Genetic enhancement of disease resistance is considered to be effective to control the disease. To provide an insight into the genomic basis of MAS disease resistance, in this study, we conducted a genome-wide association study (GWAS) to identify quantitative trait loci (QTL). A total of 1820 interspecific backcross catfish of 7 families were challenged with A. hydrophila, and 382 phenotypic extremes were selected for genotyping with the catfish 690 K SNP arrays. Three QTL on linkage group (LG) 2, 26 and 29 were identified to be significantly associated with MAS resistance. Within these regions, a total of 24 genes had known functions in immunity, 10 of which were involved in NF-κB signaling pathway, suggesting the importance of NF-κB signaling pathway in MAS resistance. In addition, three suggestively significant QTL were identified on LG 11, 17, and 20. The limited numbers of QTL involved in MAS resistance suggests that marker-assisted selection may be a viable approach for catfish breeding.

FOXO genes in channel catfish and their response after bacterial infection.

FOXO proteins are a subgroup of the forkhead family of transcription factors that play crucial roles in lifespan regulation. In addition, FOXO proteins are also involved in immune responses. After a systematic study of FOXO genes in channel catfish, Ictalurus punctatus, seven FOXO genes were identified and characterized, including FOXO1a, FOXO1b, FOXO3a, FOXO3b, FOXO4, FOXO6a and FOXO6b. Through phylogenetic and syntenic analyses, it was found that FOXO1, FOXO3 and FOXO6 were duplicated in the catfish genome, as in the zebrafish genome. Analysis of the relative rates of nonsynonymous (dN) and synonymous (dS) substitutions revealed that the FOXO genes were globally strongly constrained by negative selection. Differential expression patterns were observed in the majority of FOXO genes after Edwardsiella ictaluri and Flavobacterium columnare infections. After E. ictaluri infection, four FOXO genes with orthologs in mammal species were significantly upregulated, where FOXO6b was the most dramatically upregulated. However, after F. columnare infection, the expression levels of almost all FOXO genes were not significantly affected. These results suggested that either a pathogenesis-specific pattern or tissue-specific pattern existed in catfish after these two bacterial infections. Taken together, these findings indicated that FOXO genes may play important roles in immune responses to bacterial infections in catfish.

The Y chromosome sequence of the channel catfish suggests novel sex determination mechanisms in teleost fish.

BACKGROUND: Sex determination mechanisms in teleost fish broadly differ from mammals and birds, with sex chromosomes that are far less differentiated and recombination often occurring along the length of the X and Y chromosomes, posing major challenges for the identification of specific sex determination genes. Here, we take an innovative approach of comparative genome analysis of the genomic sequences of the X chromosome and newly sequenced Y chromosome in the channel catfish. RESULTS: Using a YY channel catfish as the sequencing template, we generated, assembled, and annotated the Y genome sequence of channel catfish. The genome sequence assembly had a contig N50 size of 2.7 Mb and a scaffold N50 size of 26.7 Mb. Genetic linkage and GWAS analyses placed the sex determination locus within a genetic distance less than 0.5 cM and physical distance of 8.9 Mb. However, comparison of the channel catfish X and Y chromosome sequences showed no sex-specific genes. Instead, comparative RNA-Seq analysis between females and males revealed exclusive sex-specific expression of an isoform of the breast cancer anti-resistance 1 (BCAR1) gene in the male during early sex differentiation. Experimental knockout of BCAR1 gene converted genetic males (XY) to phenotypic females, suggesting BCAR1 as a putative sex determination gene. CONCLUSIONS: We present the first Y chromosome sequence among teleost fish, and one of the few whole Y chromosome sequences among vertebrate species. Comparative analyses suggest that sex-specific isoform expression through alternative splicing may underlie sex determination processes in the channel catfish, and we identify BCAR1 as a potential sex determination gene.

Penaeid shrimp genome provides insights into benthic adaptation and frequent molting.

Crustacea, the subphylum of Arthropoda which dominates the aquatic environment, is of major importance in ecology and fisheries. Here we report the genome sequence of the Pacific white shrimp Litopenaeus vannamei, covering ~1.66 Gb (scaffold N50 605.56 Kb) with 25,596 protein-coding genes and a high proportion of simple sequence repeats (>23.93%). The expansion of genes related to vision and locomotion is probably central to its benthic adaptation. Frequent molting of the shrimp may be explained by an intensified ecdysone signal pathway through gene expansion and positive selection. As an important aquaculture organism, L. vannamei has been subjected to high selection pressure during the past 30 years of breeding, and this has had a considerable impact on its genome. Decoding the L. vannamei genome not only provides an insight into the genetic underpinnings of specific biological processes, but also provides valuable information for enhancing crustacean aquaculture.

Heat stress induced alternative splicing in catfish as determined by transcriptome analysis.

Heat tolerance is increasingly becoming an important trait for aquaculture species with a changing climate. Transcriptional studies on responses to heat stress have been conducted in catfish, one of the most important economic aquaculture species around the world. The molecular mechanisms underlying heat tolerance is still poorly understood, especially at the post-transcriptional level including regulation of alternative splicing. In this study, existing RNA-Seq datasets were utilized to characterize the change of alternative splicing in catfish following heat treatment. Heat-tolerant and -intolerant catfish were differentiated by the time to lost equilibrium after heat stress. With heat stress, alternative splicing was generally increased. In heat-intolerant fish, the thermal stress induced 29.2% increases in alternative splicing events and 25.8% increases in alternatively spliced genes. A total of 282, 189, and 44 differential alternative splicing (DAS) events were identified in control-intolerant, control-tolerant, and intolerant-tolerant comparisons, corresponding to 252, 171, and 42 genes, respectively. Gene ontology analyses showed that genes involved in the molecular function of RNA binding were significantly enriched in DAS gene sets after heat stress in both heat-intolerant and -tolerant catfish compared with the control group. Similar results were also observed in the DAS genes between heat-intolerant and -tolerant catfish, and the biological process of RNA splicing was also enriched in this comparison, indicating the involvement of RNA splicing-related genes underlying heat tolerance. This is the first comprehensive study of alternative splicing in response to heat stress in fish species, providing insights into the molecular mechanisms of responses to the abiotic stress.