Hypoxia enhances autophagy level of human sperms.

The relationship between oxygen sensing and autophagy in human sperms was explored in this study. Health semen and asthenozoospermia (astheno) semen were incubated with hypoxia-inducible factor-1α (HIF-1α) interferents, i.e., lificiguat (YC-1) or cobalt chloride (CoCl2), respectively. Label-free quantitative proteomic technology was used to identify the differentially expressed proteins in human semen under the hypoxia condition. Selected proteins were detected with ELISA. It was found that the autophagy levels of sperm in the YC-1 + health group or CoCl2 + astheno group increased while the vitality decreased. A total of 17, 34 and 35 differentially expressed proteins were observed in the Astheno group, the YC-1 + health group and the CoCl2 + astheno group, respectively. These proteins were primarily associated with protein processing in endoplasmic reticulum, Th17 cell differentiation, progesterone-mediated oocyte maturation, glycolysis/gluconeogenesis, HIF-1 signaling pathway, biosynthesis of amino acids, and carbon metabolism. The expression levels of protein HIF-1α, LC3B, histone H4, cathepsin L and ENO1 changed significantly in the groups. The study suggests that hypoxia can increase sperm autophagy level and reduce their vitality through HIF-1 signaling pathway and glycolysis/gluconeogenesis signaling pathway. Furthermore, proteins histone H4, cathepsin L, glutathione synthetase and ENO1 are proposed as potential biomarkers of autophagy and vitality in asthenozoospermia sperm.

Recent Development of Versatile Polyphenol Platforms in Fertilizers and Pesticides.

The utilization of agrochemicals has been of significant importance in both the cultivation and disease control of crops. The development of advanced agrochemicals that are both effective and eco-friendly has been made possible through the use of slow delivery platforms and surface modification technology. Inspired by the nature of mussel adhesion, polyphenolic platforms with versatile properties have been extensively employed in various applications, including agro-food, owing to their ability to flexibly modulate chemical and surface characteristics. This mini-review highlights the development of polyphenols, such as polydopamine and tannic acid, in the field of agrochemicals, particularly in the design and production of novel fertilizers and pesticides. The synthetic approach, active ingredient release performance, foliar adhesion, and design of polyphenolic-based agrochemicals in recent years have been discussed to explore their potential applications and limitations. We believe that utilizing versatile polyphenolic materials and their characteristics for agro-food applications can provide innovative ideas and suggestions for developing novel agrochemicals suitable for modern and sustainable horticulture and agriculture.

Genome assembly and genetic dissection of a prominent drought-resistant maize germplasm.

In the context of climate change, drought is one of the most limiting factors that influence crop production. Maize, as a major crop, is highly vulnerable to water deficit, which causes significant yield loss. Thus, identification and utilization of drought-resistant germplasm are crucial for the genetic improvement of the trait. Here we report on a high-quality genome assembly of a prominent drought-resistant genotype, CIMBL55. Genomic and genetic variation analyses revealed that 65 favorable alleles of 108 previously identified drought-resistant candidate genes were found in CIMBL55, which may constitute the genetic basis for its excellent drought resistance. Notably, ZmRtn16, encoding a reticulon-like protein, was found to contribute to drought resistance by facilitating the vacuole H+-ATPase activity, which highlights the role of vacuole proton pumps in maize drought resistance. The assembled CIMBL55 genome provided a basis for genetic dissection and improvement of plant drought resistance, in support of global food security.

Visible-Light-Driven Zinc Oxide Quantum Dots for the Management of Bacterial Fruit Blotch Disease and the Improvement of Melon Seedlings Growth.

Bacterial fruit blotch is one of the most destructing diseases of melon producing-regions. Here, zinc oxide quantum dots (ZnO QDs) were synthesized, and their antibacterial activity against Acidovorax citrulli was investigated. The results indicated that the obtained ZnO QDs displayed 5.7-fold higher antibacterial activity than a commercial Zn-based bactericide (zinc thiazole). Interestingly, the antibacterial activity of ZnO QDs irradiated with light was 1.8 times higher than that of the dark-treated group. It was because ZnO QDs could induce the generation of hydroxyl radicals and then up-regulate the expression of oxidative stress-related genes, finally leading to the loss of cell membrane integrity. A pot experiment demonstrated that foliar application of ZnO QDs significantly reduced the bacterial fruit blotch disease incidence (32.0%). Furthermore, the supply of ZnO QDs could improve the growth of infected melon seedlings by activating the antioxidant defense system. This work provides a promising light-activated quantum-bactericide for the management of pathogenic bacterial infections in melon crop protection.

Natural variations of ZmSRO1d modulate the trade-off between drought resistance and yield by affecting ZmRBOHC-mediated stomatal ROS production in maize.

While crop yields have historically increased, drought resistance has become a major concern in the context of global climate change. The trade-off between crop yield and drought resistance is a common phenomenon; however, the underlying molecular modulators remain undetermined. Through genome-wide association study, we revealed that three non-synonymous variants in a drought-resistant allele of ZmSRO1d-R resulted in plasma membrane localization and enhanced mono-ADP-ribosyltransferase activity of ZmSRO1d toward ZmRBOHC, which increased reactive oxygen species (ROS) levels in guard cells and promoted stomatal closure. ZmSRO1d-R enhanced plant drought resilience and protected grain yields under drought conditions, but it led to yield drag under favorable conditions. In contrast, loss-of-function mutants of ZmRBOHC showed remarkably increased yields under well-watered conditions, whereas they showed compromised drought resistance. Interestingly, by analyzing 189 teosinte accessions, we found that the ZmSRO1d-R allele was present in teosinte but was selected against during maize domestication and modern breeding. Collectively, our work suggests that the allele frequency reduction of ZmSRO1d-R in breeding programs may have compromised maize drought resistance while increased yields. Therefore, introduction of the ZmSRO1d-R allele into modern maize cultivars would contribute to food security under drought stress caused by global climate change.

Genome-Wide Association Analyses to Identify SNPs Related to Drought Tolerance.

Drought stress is a serious agronomic problem resulting in significant yield losses globally. Breeding cultivars with drought tolerance is an important strategy that can be used to address this problem. Drought tolerance, however, is a complex multigenic trait, making advancements with conventional breeding approaches very challenging. This emphasizes the importance of dissecting the genetics of this trait and the identification and cloning of genes responsible for drought tolerance. With the rapid development of sequencing technologies and analytic methodologies, genome-wide association study (GWAS) has become an important tool for detecting natural variations underlying complex traits in crops. Identified loci can serve as targets for genomic selection or precise editing that enables the molecular design of new cultivars. This chapter describes the pipeline of statistical methods used in GWAS analysis, and covers field design, quality control, population structure control, association tests, and visualization of data. GWAS methodology used to dissect the genetic basis of drought tolerance is presented, and perspectives for optimizing the design and analysis of GWAS are discussed. The provided information serves as a valuable resource for researchers interested in GWAS technology.

Co-Expression of ZmVPP1 with ZmNAC111 Confers Robust Drought Resistance in Maize.

Drought is a primary environmental factor limiting maize production globally. Although transferring a single gene to maize can enhance drought resistance, maize response to water deficit requires further improvement to accommodate the steadily intensifying drought events worldwide. Here, we generated dual transgene lines simultaneously overexpressing two drought-resistant genes, ZmVPP1 (encoding a vacuolar-type H+ pyrophosphatase) and ZmNAC111 (encoding a NAM, ATAF, and CUC (NAC)-type transcription factor). Following drought stress, survival rates of the pyramided transgenic seedlings reached 62-66%, while wild-type and single transgene seedling survival rates were 23% and 37-42%, respectively. Maize seedlings co-expressing ZmVPP1 and ZmNAC111 exhibited higher photosynthesis rates, antioxidant enzyme activities, and root-shoot ratios than the wild type, and anthesis-silking intervals were shorter while grain yields were higher under water deficit conditions in field trials. Additionally, RNA-sequencing analysis confirmed that photosynthesis and stress-related metabolic processes were stimulated in the dual transgene plants under drought conditions. The findings in this work illustrate how high co-expression of different drought-related genes can reinforce drought resistance over that of individual transgene lines, providing a path for developing arid climate-adapted elite maize varieties.

Genetic dissection of maize drought tolerance for trait improvement.

Maize is one of the most important crops, but its production is threatened by drought stress worldwide. Thus, increased drought tolerance has been a major goal of maize breeding. Conventional breeding strategies have led to significantly increase of maize yields; however, these strategies often fail to meet the need for drought stress tolerance enhancement. Here, we focus on progress related to the genetic dissection of drought tolerance in maize at different developmental stages achieved through linkage mapping and association mapping. Moreover, recent molecular breeding systems, including transgenic, genome-wide marker-assisted selection, and genome editing technologies, have provided a more direct, efficient, and accurate approach for trait improvement. We also provide perspectives on future directions regarding multi-omics studies and maize improvement. Overall, the application of acquired knowledge will facilitate maize breeding to meet the challenges.

Characteristics of plant growth-promoting rhizobacteria SCPG-7 and its effect on the growth of Capsicum annuum L.

The strain SCPG-7 was isolated from saline soil in a cotton field. It is confirmed that the strain SCPG-7 is Pseudomonas sp. by means of the analysis of its phenotypic features and 16S rRNA sequence. SCPG-7 was capable of dissolving mineral tri-calcium phosphate (Ca3(PO4)2) and tri-magnesium phosphate (Mg3(PO4)2). In contrast, no showing iron phosphate (FePO4) or aluminum phosphate (AlPO4) solubilizing activities were detected by this experimental approach. The ratio of the dissolved P diameter to the colony diameter was 1.86. To study the phosphate dissolving mechanisms of the strain, we analyzed the changes of the pH value, the soluble phosphate content, the concentration of alkaline phosphatase, and the production of organic acid in the insoluble phosphate liquid medium. 2-keto-D-gluconicacid, α-ketoglutaric acid, succinic acid, etc. were characterized by LC-MS/MS in NBRIP medium. The concentration of 2-keto-D-gluconicacid increased to 88.6 mg/L after being cultured for 216 h. The strain decreased the pH value of the medium from 7.4 to 4.7 and the released soluble phosphate up to 516 mg/L, which proved the production of organic acids and alkaline phosphatase to be mechanism for solubilizing P. Under low phosphorus stress, Pseudomonas global regulatory protein PhoB regulates the transcription of the alkaline phosphatase gene. IAA and siderophore were secreted by SCPG-7. After treatment with SCPG-7, the individual plant height and dry weight of pepper increased by 23.3 and 31.2%, respectively, compared to the control group. The results show that the strain SCPG-7 has the potential to convert insoluble inorganic phosphorus to plant-available phosphorus. It can enhance soil phosphorus release through biological pathways, thereby increasing crop yield, and providing germplasm resources for the development of biological fertilizers.

Mapping regulatory variants controlling gene expression in drought response and tolerance in maize.

BACKGROUND: Gene expression is a key determinant of cellular response. Natural variation in gene expression bridges genetic variation to phenotypic alteration. Identification of the regulatory variants controlling the gene expression in response to drought, a major environmental threat of crop production worldwide, is of great value for drought-tolerant gene identification. RESULTS: A total of 627 RNA-seq analyses are performed for 224 maize accessions which represent a wide genetic diversity under three water regimes; 73,573 eQTLs are detected for about 30,000 expressing genes with high-density genome-wide single nucleotide polymorphisms, reflecting a comprehensive and dynamic genetic architecture of gene expression in response to drought. The regulatory variants controlling the gene expression constitutively or drought-dynamically are unraveled. Focusing on dynamic regulatory variants resolved to genes encoding transcription factors, a drought-responsive network reflecting a hierarchy of transcription factors and their target genes is built. Moreover, 97 genes are prioritized to associate with drought tolerance due to their expression variations through the Mendelian randomization analysis. One of the candidate genes, Abscisic acid 8'-hydroxylase, is verified to play a negative role in plant drought tolerance. CONCLUSIONS: This study unravels the effects of genetic variants on gene expression dynamics in drought response which allows us to better understand the role of distal and proximal genetic effects on gene expression and phenotypic plasticity. The prioritized drought-associated genes may serve as direct targets for functional investigation or allelic mining.

Genetic variation in ZmTIP1 contributes to root hair elongation and drought tolerance in maize.

Drought is a major abiotic stress that threatens maize production globally. A previous genome-wide association study identified a significant association between the natural variation of ZmTIP1 and the drought tolerance of maize seedlings. Here, we report on comprehensive genetic and functional analysis, indicating that ZmTIP1, which encodes a functional S-acyltransferase, plays a positive role in regulating the length of root hairs and the level of drought tolerance in maize. We show that enhancing ZmTIP1 expression in transgenic Arabidopsis and maize increased root hair length, as well as plant tolerance to water deficit. In contrast, ZmTIP1 transposon-insertional mutants displayed the opposite phenotype. A calcium-dependent protein kinase, ZmCPK9, was identified as a substrate protein of ZmTIP1, and ZmTIP1-mediated palmitoylation of two cysteine residues facilitated the ZmCPK9 PM association. The results of this research enrich our knowledge about ZmTIP1-mediated protein S-acylation modifications in relation to the regulation of root hair elongation and drought tolerance. Additionally, the identification of a favourable allele of ZmTIP1 also provides a valuable genetic resource or selection target for the genetic improvement of maize.

Characterization of Proteome Variation During Modern Maize Breeding.

The success of modern maize breeding has been demonstrated by remarkable increases in productivity with tremendous modification of agricultural phenotypes over the last century. Although the underlying genetic changes of the maize adaptation from tropical to temperate regions have been extensively studied, our knowledge is limited regarding the accordance of protein and mRNA expression levels accompanying such adaptation. Here we conducted an integrative analysis of proteomic and transcriptomic changes in a maize association panel. The minimum extent of correlation between protein and RNA levels suggests that variation in mRNA expression is often not indicative of protein expression at a population scale. This is corroborated by the observation that mRNA- and protein-based coexpression networks are relatively independent of each other, and many pQTLs arise without the presence of corresponding eQTLs. Importantly, compared with transcriptome, the subtypes categorized by the proteome show a markedly high accuracy to resemble the genomic subpopulation. These findings suggest that proteome evolved under a greater evolutionary constraint than transcriptome during maize adaptation from tropical to temperate regions. Overall, the integrated multi-omics analysis provides a functional context to interpret gene expression variation during modern maize breeding.

Genetic variation in ZmVPP1 contributes to drought tolerance in maize seedlings.

Maize production is threatened by drought stress worldwide. Identification of the genetic components underlying drought tolerance in maize is of great importance. Here we report a genome-wide association study (GWAS) of maize drought tolerance at the seedling stage that identified 83 genetic variants, which were resolved to 42 candidate genes. The peak GWAS signal showed that the natural variation in ZmVPP1, encoding a vacuolar-type H(+) pyrophosphatase, contributes most significantly to the trait. Further analysis showed that a 366-bp insertion in the promoter, containing three MYB cis elements, confers drought-inducible expression of ZmVPP1 in drought-tolerant genotypes. Transgenic maize with enhanced ZmVPP1 expression exhibits improved drought tolerance that is most likely due to enhanced photosynthetic efficiency and root development. Taken together, this information provides important genetic insights into the natural variation of maize drought tolerance. The identified loci or genes can serve as direct targets for both genetic engineering and selection for maize trait improvement.

[Expression of miR-429 and Its Target Gene HSPA4L in Sperms from Asthenospermia Patients.]

OBJECTIVES: To investigate the expression of miR-429 and its target gene heat shock protein A4L (HSPA4L) in sperms from asthenospermia patients. METHODS: Twenty semen samples from healthy and fertile adults and 20 semen samples from asthenospermia patients were collected,and normal sperm parameters were defined according to World Health Organization criteria.The expression levels of miR-429 and HSPA4L mRNA were determined by qRT-PCR,and the bioinformatics tool (Targetscan) was used to predict the target of miR-429.Luciferase reporter assay and transfection study were performed to confirm target gene of miR-429.The expression levels of HSPA4L mRNA and protein were further determined by qRT-CPR and Western blot,respectively. RESULTS: The motility and viability of sperms from asthenospermia patients were lower than that in control group,and miR-429 was up-regulated in sperms from asthenospermia patients.Bioinformatics analysis revealed that HSPA4L was a target of miR-429.Luciferase reporter assay and transfection study further confirmed that miR-429 suppresses the expressions of HSPA4L mRNA and protein via directly targeting HSPA4L 3'UTR.Results from clinical samples also demonstrated that HSPA4L mRNA and protein were down-regulated in sperms from asthenospermia patients and the expression level of miR-429 was inversely correlated with the expression level of HSPA4L mRNA (r=-0.725, P<0.05). CONCLUSIONS: miR-429 is up-regulated in sperms from asthenospermia patients,and it may modulate the motility and viability of sperms via suppressing the expression of HSPA4L.

A transposable element in a NAC gene is associated with drought tolerance in maize seedlings.

Drought represents a major constraint on maize production worldwide. Understanding the genetic basis for natural variation in drought tolerance of maize may facilitate efforts to improve this trait in cultivated germplasm. Here, using a genome-wide association study, we show that a miniature inverted-repeat transposable element (MITE) inserted in the promoter of a NAC gene (ZmNAC111) is significantly associated with natural variation in maize drought tolerance. The 82-bp MITE represses ZmNAC111 expression via RNA-directed DNA methylation and H3K9 dimethylation when heterologously expressed in Arabidopsis. Increasing ZmNAC111 expression in transgenic maize enhances drought tolerance at the seedling stage, improves water-use efficiency and induces upregulation of drought-responsive genes under water stress. The MITE insertion in the ZmNAC111 promoter appears to have occurred after maize domestication and spread among temperate germplasm. The identification of this MITE insertion provides insight into the genetic basis for natural variation in maize drought tolerance.

The complete genome sequence of a novel mycovirus from Alternaria longipes strain HN28.

The complete nucleotide sequence of Alternaria longipes dsRNA virus 1 (AlRV1), a novel double-stranded RNA (dsRNA) mycovirus, was determined and analyzed. AlRV1-HN28 contains a single dsRNA genome segment 3415 base pairs in length (excluding the 3' poly(A) tail) and was predicted to contain two discontiguous open reading frames (ORFs, ORF A and ORF B). The 5'-proximal ORF A (1182 nt) potentially encodes a protein of 394 amino acids (aa) with a predicted molecular mass of 43 kDa; this protein showed no significant similarities to any other sequences in any of the NCBI protein databases. The 3'-proximal ORF B (1737 nt) encodes a protein of 579 aa with a predicted molecular mass of 65 kDa; this protein sequence shares similarities with the conserved domains of RNA-dependent RNA polymerases of other mycoviruses. Phylogenetic analysis indicated that AlRV1-HN28 was closely related to four other unclassified viruses, which suggests that the AlRV1-HN28 isolated from Alternaria longipes may belong to a new family of dsRNA mycoviruses. This is the first report of the full-length nucleotide sequence of a mycovirus that infects Alternaria longipes.

A novel single-stranded RNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix, with similarity to hypo-like viruses.

Here we report a biological and molecular characterization of a novel positive-sense RNA virus isolated from a field isolate (NW10) of a filamentous phytopathogenic fungus, the white root rot fungus that is designated as Rosellinia necatrix fusarivirus 1 (RnFV1). A recently developed technology using zinc ions allowed us to transfer RnFV1 to two mycelially incompatible Rosellinia necatrix strains. A biological comparison of the virus-free and -recipient isogenic fungal strains suggested that RnFV1 infects latently and thus has no potential as a virocontrol agent. The virus has an undivided positive-sense RNA genome of 6286 nucleotides excluding a poly (A) tail. The genome possesses two non-overlapping open reading frames (ORFs): a large ORF1 that encodes polypeptides with RNA replication functions and a smaller ORF2 that encodes polypeptides of unknown function. A lack of coat protein genes was suggested by the failure of virus particles from infected mycelia. No evidence was obtained by Northern analysis or classical 5'-RACE for the presence of subgenomic RNA for the downstream ORF. Sequence similarities were found in amino-acid sequence between RnFV1 putative proteins and counterparts of a previously reported mycovirus, Fusarium graminearum virus 1 (FgV1). Interestingly, several related sequences were detected by BLAST searches of independent transcriptome assembly databases one of which probably represents an entire virus genome. Phylogenetic analysis based on the conserved RNA-dependent RNA polymerase showed that RnFV1, FgV1, and these similar sequences are grouped in a cluster distinct from distantly related hypoviruses. It is proposed that a new taxonomic family termed Fusariviridae be created to include RnFV1 and FgV1.

Genome-wide analysis of ZmDREB genes and their association with natural variation in drought tolerance at seedling stage of Zea mays L.

The worldwide production of maize (Zea mays L.) is frequently impacted by water scarcity and as a result, increased drought tolerance is a priority target in maize breeding programs. While DREB transcription factors have been demonstrated to play a central role in desiccation tolerance, whether or not natural sequence variations in these genes are associated with the phenotypic variability of this trait is largely unknown. In the present study, eighteen ZmDREB genes present in the maize B73 genome were cloned and systematically analyzed to determine their phylogenetic relationship, synteny with rice, maize and sorghum genomes; pattern of drought-responsive gene expression, and protein transactivation activity. Importantly, the association between the nucleic acid variation of each ZmDREB gene with drought tolerance was evaluated using a diverse population of maize consisting of 368 varieties from tropical and temperate regions. A significant association between the genetic variation of ZmDREB2.7 and drought tolerance at seedling stage was identified. Further analysis found that the DNA polymorphisms in the promoter region of ZmDREB2.7, but not the protein coding region itself, was associated with different levels of drought tolerance among maize varieties, likely due to distinct patterns of gene expression in response to drought stress. In vitro, protein-DNA binding assay demonstrated that ZmDREB2.7 protein could specifically interact with the target DNA sequences. The transgenic Arabidopsis overexpressing ZmDREB2.7 displayed enhanced tolerance to drought stress. Moreover, a favorable allele of ZmDREB2.7, identified in the drought-tolerant maize varieties, was effective in imparting plant tolerance to drought stress. Based upon these findings, we conclude that natural variation in the promoter of ZmDREB2.7 contributes to maize drought tolerance, and that the gene and its favorable allele may be an important genetic resource for the genetic improvement of drought tolerance in maize.

Cultivated grapevines represent a symptomless reservoir for the transmission of hop stunt viroid to hop crops: 15 years of evolutionary analysis.

Hop stunt was a mysterious disorder that first emerged in the 1940s in commercial hops in Japan. To investigate the origin of this disorder, we infected hops with natural Hop stunt viroid (HpSVd) isolates derived from four host species (hop, grapevine, plum and citrus), which except for hop represent possible sources of the ancestral viroid. These plants were maintained for 15 years, then analyzed the HpSVd variants present. Here we show that the variant originally found in cultivated grapevines gave rise to various combinations of mutations at positions 25, 26, 54, 193, and 281. However, upon prolonged infection, these variants underwent convergent evolution resulting in a limited number of adapted mutants. Some of them showed nucleotide sequences identical to those currently responsible for hop stunt epidemics in commercial hops in Japan, China, and the United States. Therefore, these results indicate that we have successfully reproduced the original process by which a natural HpSVd variant naturally introduced into cultivated hops was able to mutate into the HpSVd variants that are currently present in commercial hops. Furthermore, and importantly, we have identified cultivated grapevines as a symptomless reservoir in which HSVd can evolve and be transmitted to hop crops to cause epidemics.

[Effects of salicylic acid on the leaf photosynthesis and antioxidant enzyme activities of cucumber seedlings under low temperature and light intensity].

In order to elucidate the regulation functions of salicylic acid (SA) on the photosynthesis of cucumber under low temperature and light intensity, the seedlings of cucumber 'Jinyou 3' under low temperature and light intensity were foliar-sprayed with different concentration SA, and the leaf gas exchange parameters, photochemical efficiency, MDA content, and antioxidant enzyme activities were measured. The results showed that under low temperature and light intensity, the leaf photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), actual photochemical efficiency of PS II (PhiPSII), and maximal photochemical efficiency of PS II (Fv/Fm) of the seedlings all decreased but the intercellular CO2 concentration (Ci) increased, suggesting that nonstomatal limitation was the main cause of the decrease of Pn under low temperature and light intensity stress. Low temperature and light intensity also led to the increase of leaf malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, the decrease of catalase (CAT) activity, and the decrease after an initial increase of peroxidase (POD) activity. However, foliar-spraying 0.5-2.5 mmol x L(-1) of SA before the stress increased the leaf Pn, Gs, Tr, PhiPSII, Fv/Fm, and activities of SOD, POD and CAT while decreased the Ci and MDA content, suggesting that SA could regulate the leaf photosynthetic functions of cucumber seedlings, and enhance the seedlings resistance against low temperature and light intensity. The optimum concentration of SA for the foliar-spraying was 1 mmol x L(-1).