Utility of Arabidopsis KASII Promoter in Development of an Effective CRISPR/Cas9 System for Soybean Genome Editing and Its Application in Engineering of Soybean Seeds Producing Super-High Oleic and Low Saturated Oils.

This study reports the use of the Arabidopsis KASII promoter (AtKASII) to develop an efficient CRISPR/Cas9 system for soybean genome editing. When this promoter was paired with Arabidopsis U6 promoters to drive Cas9 and single guide RNA expression, respectively, simultaneous editing of the three fatty acid desaturase genes GmFAD2-1A, GmFAD2-1B, and GmFAD3A occurred in more than 60% of transgenic soybean lines at T2 generation, and all the triple mutants possessed desirable high-oleic traits. In sharp contrast, not a single line underwent simultaneous editing of the three target genes when AtKASII was replaced by the widely used AtEC1.2 promoter. Furthermore, our study showed that the stable and inheritable mutations in the high-oleic lines did not alter the overall contents of oil and protein or amino acid composition while increasing the oleic acid content up to 87.6% from approximately 23.8% for wild-type seeds, concomitant with 34.4- and 3.7-fold reductions in linoleic and linolenic acid, respectively. Collectively, this study demonstrates that the AtKASII promoter is highly promising for optimization of the CRISPR/Cas9 system for genome editing in soybean and possibly beyond.

Genome-wide identification and expression of monoacylglycerol lipase (MAGL) gene family in peanut (Arachis hypogaea L.) and functional analysis of AhMGATs in neutral lipid metabolism.

Monoacylglycerol lipase (MAGL) involved in regulating plant growth and development and stress responses, hydrolyzes monoacylglycerol (MAG) into free fatty acid and glycerol, which is the last step of triacylglycerol (TAG) breakdown. Here, a genome-wide characterization of MAGL gene family from cultivated peanut (Arachis hypogaea L.) was performed. In total, 24 MAGL genes were identified and unevenly distributed on 14 chromosomes, encoding 229-414 amino acids with molecular weights ranging from 25.91 to 47.01 kDa. Spatiotemporal and stress-induced expression was analyzed by qRT-PCR. Multiple sequence alignment revealed that AhMAGL1a/b and AhMAGL3a/b were the only four bifunctional enzymes with conserved regions of hydrolase and acyltransferase, which could also be named as AhMGATs. GUS histochemical assay showed that AhMAGL1a and -1b were strongly expressed in all tissues of the plants; whereas both AhMAGL3a and -3b were weakly expressed in plants. Subcellular localization analysis indicated that AhMGATs were localized in the endoplasmic reticulum and/or Golgi complex. Seed-specific overexpression of AhMGATs in Arabidopsis decreased the oil content of the seeds and altered the fatty acid compositions, indicating that AhMGATs were involved in TAG breakdown but not TAG biosynthesis in plant seeds. This study lays the foundation for better understanding AhMAGL genes biological function in planta.

Ectopic Expression of AeNAC83, a NAC Transcription Factor from Abelmoschus esculentus, Inhibits Growth and Confers Tolerance to Salt Stress in Arabidopsis.

NAC transcription factors play crucial roles in plant growth, development and stress responses. Previously, we preliminarily identified that the transcription factor AeNAC83 gene was significantly up-regulated under salt stress in okra (Abelmoschus esculentus). Herein, we cloned the nuclear-localized AeNAC83 from okra and identified its possible role in salt stress response and plant growth. The down-regulation of AeNAC83 caused by virus-induced gene silencing enhanced plant sensitivity to salt stress and increased the biomass accumulation of okra seedlings. Meanwhile, AeNAC83-overexpression Arabidopsis lines improved salt tolerance and exhibited many altered phenotypes, including small rosette, short primary roots, and promoted crown roots and root hairs. RNA-seq showed numerous genes at the transcriptional level that changed significantly in the AeNAC83-overexpression transgenic and the wild Arabidopsis with or without NaCl treatment, respectively. The expression of most phenylpropanoid and flavonoid biosynthesis-related genes was largely induced by salt stress. While genes encoding key proteins involved in photosynthesis were almost declined dramatically in AeNAC83-overexpression transgenic plants, and NaCl treatment further resulted in the down-regulation of these genes. Furthermore, DEGs encoding various plant hormone signal pathways were also identified. These results indicate that AeNAC83 is involved in resistance to salt stress and plant growth.

Comparative physiological and full-length transcriptome analyses reveal the molecular mechanism of melatonin-mediated salt tolerance in okra (Abelmoschus esculentus L.).

BACKGROUND: Melatonin, a multifunctional signal molecule, has been reported to play crucial roles in growth and development and stress responses in various plant species. Okra (Abelmoschus esculentus L.) is a food crop with extremely high values of nutrition and healthcare. Recent reports have revealed the protective role of melatonin in alleviating salt stress. However, little is known about its regulatory mechanisms in response to salt stress in okra. RESULTS: In this study, we explored whether exogenous melatonin pretreatment could alleviate salt stress (300 mM NaCl) of okra plants. Results showed that exogenous application of melatonin (50 µM) significantly enhanced plant tolerance to salt stress, as demonstrated by the plant resistant phenotype, as well as by the higher levels of the net photosynthetic rate, chlorophyll fluorescence and chlorophyll content in comparison with nontreated salt-stressed plants. Additionally, melatonin pretreatment remarkably decreased the levels of lipid peroxidation and H2O2 content and scavenged O2•- in melatonin-pretreated plants, which may be attributed to the higher levels of enzyme activities including POD and GR. Moreover, a combination of third- (PacBio) and second-generation (Illumina) sequencing technologies was applied to sequence full-length transcriptomes of okra. A total of 121,360 unigenes was obtained, and the size of transcript lengths ranged from 500 to 6000 bp. Illumina RNA-seq analysis showed that: Comparing with control, 1776, 1063 and 1074 differential expression genes (DEGs) were identified from the three treatments (NaCl, MT50 and MT + NaCl, respectively). These genes were enriched in more than 10 GO terms and 34 KEGG pathways. Nitrogen metabolism, sulfur metabolism, and alanine, aspartate and glutamate metabolism were significantly enriched in all three treatments. Many transcription factors including MYB, WRKY, NAC etc., were also identified as DEGs. CONCLUSIONS: Our preliminary results suggested that melatonin pretreatment enhanced salt tolerance of okra plants for the first time. These data provide the first set of full-length isoforms in okra and more comprehensive insights into the molecular mechanism of melatonin responses to salt stress.

OsCpn60ß1 is Essential for Chloroplast Development in Rice (Oryza sativa L.).

The chaperonin 60 (Cpn60) protein is of great importance to plants due to its involvement in modulating the folding of numerous chloroplast protein polypeptides. In chloroplasts, Cpn60 is differentiated into two subunit types-Cpn60α and Cpn60ß and the rice genome encodes three α and three ß plastid chaperonin subunits. However, the functions of Cpn60 family members in rice were poorly understood. In order to investigate the molecular mechanism of OsCpn60ß1, we attempted to disrupt the OsCpn60ß1 gene by CRISPR/Cas9-mediated targeted mutagenesis in this study. We succeeded in the production of homozygous OsCpn60ß1 knockout rice plants. The OsCpn60ß1 mutant displayed a striking albino leaf phenotype and was seedling lethal. Electron microscopy observation demonstrated that chloroplasts were severely disrupted in the OsCpn60ß1 mutant. In addition, OsCpn60ß1 was located in the chloroplast and OsCpn60ß1 is constitutively expressed in various tissues particularly in the green tissues. The label-free qualitative proteomics showed that photosynthesis-related pathways and ribosomal pathways were significantly inhibited in OsCpn60ß1 mutants. These results indicate that OsCpn60ß1 is essential for chloroplast development in rice.

Hyperspectral imaging combined with machine learning as a tool to obtain high-throughput plant salt-stress phenotyping.

The rapid selection of salinity-tolerant crops to increase food production in salinized lands is important for sustainable agriculture. Recently, high-throughput plant phenotyping technologies have been adopted that use plant morphological and physiological measurements in a non-destructive manner to accelerate plant breeding processes. Here, a hyperspectral imaging (HSI) technique was implemented to monitor the plant phenotypes of 13 okra (Abelmoschus esculentus L.) genotypes after 2 and 7 days of salt treatment. Physiological and biochemical traits, such as fresh weight, SPAD, elemental contents and photosynthesis-related parameters, which require laborious, time-consuming measurements, were also investigated. Traditional laboratory-based methods indicated the diverse performance levels of different okra genotypes in response to salinity stress. We introduced improved plant and leaf segmentation approaches to RGB images extracted from HSI imaging based on deep learning. The state-of-the-art performance of the deep-learning approach for segmentation resulted in an intersection over union score of 0.94 for plant segmentation and a symmetric best dice score of 85.4 for leaf segmentation. Moreover, deleterious effects of salinity affected the physiological and biochemical processes of okra, which resulted in substantial changes in the spectral information. Four sample predictions were constructed based on the spectral data, with correlation coefficients of 0.835, 0.704, 0.609 and 0.588 for SPAD, sodium concentration, photosynthetic rate and transpiration rate, respectively. The results confirmed the usefulness of high-throughput phenotyping for studying plant salinity stress using a combination of HSI and deep-learning approaches.

Comparative proteomic analysis of okra (Abelmoschus esculentus L.) seedlings under salt stress.

BACKGROUND: Salinization seriously threatens land use efficiency and crop yields across the world. Understanding the mechanisms plants use to protect against salt stress will help breeders develop salt-tolerant vegetable crops. Okra (Abelmoschus esculentus L.) is an important vegetable crop of the mallow family, which is now cultivated in warm regions worldwide. To understand the effects of salt stress on the protein level of okra, a comparative proteomic analysis of okra seedlings grown in the presence of 0 or 300 mmol L- 1 NaCl treatment was performed using an integrated approach of Tandem Mass Tag labeling and LC-MS/MS integrated approach. RESULTS: A total of 7179 proteins were identified in this study, for which quantitative information was available for 5774 proteins. In the NaCl/control comparison group, there were 317 differentially expressed proteins (DEPs), of which 165 proteins were upregulated and 152 proteins downregulated in the presence of NaCl. Based on the above data, we carried out a systematic bioinformatics analysis of proteins with information, including protein annotation, domain characteristics, functional classification, and pathway enrichment. Enriched gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the DEPs were most strongly associated with "response to stress" and "protein processing in endoplasmic reticulum". Furthermore, several heat shock proteins were identified as DEPs. CONCLUSIONS: This information provides a reference direction for further research on the okra proteome in the downstream of the salt stress response, with our data revealing that the responses of okra to salt stress involves by various pathways.

The Phosphoproteomic Response of Okra (Abelmoschus esculentus L.) Seedlings to Salt Stress.

Soil salinization is a major environmental stresses that seriously threatens land use efficiency and crop yields worldwide. Although the overall response of plants to NaCl has been well studied, the contribution of protein phosphorylation to the detoxification and tolerance of NaCl in okra (Abelmoschus esculentus L.) seedlings is unclear. The molecular bases of okra seedlings' responses to 300 mM NaCl stress are discussed in this study. Using a combination of affinity enrichment, tandem mass tag (TMT) labeling and high-performance liquid chromatography⁻tandem mass spectrometry analysis, a large-scale phosphoproteome analysis was performed in okra. A total of 4341 phosphorylation sites were identified on 2550 proteins, of which 3453 sites of 2268 proteins provided quantitative information. We found that 91 sites were upregulated and 307 sites were downregulated in the NaCl/control comparison group. Subsequently, we performed a systematic bioinformatics analysis including gene ontology annotation, domain annotation, subcellular localization, and Kyoto Encyclopedia of Genes and Genomes pathway annotation. The latter revealed that the differentially expressed proteins were most strongly associated with 'photosynthesis antenna proteins' and 'RNA degradation'. These differentially expressed proteins probably play important roles in salt stress responses in okra. The results should help to increase our understanding of the molecular mechanisms of plant post-translational modifications in response to salt stress.

Correction: Transcriptome analysis of tomato (Solanum lycopersicum L.) shoots reveals a crosstalk between auxin and strigolactone.

[This corrects the article DOI: 10.1371/journal.pone.0201124.].

Transcriptome analysis of tomato (Solanum lycopersicum L.) shoots reveals a crosstalk between auxin and strigolactone.

Auxin and strigolactone (SL) are two important phytohormones involved in shoot branching and morphology. Tomato (Solanum lycopersicum L.), a member of the Solanaceae family, is one of the most popular food crops with high economic value in the world. To seek a better understanding of the responses to exogenous hormones, transcriptome analyses of the tomato shoots treated with exogenous auxin and SL, separately or together, were performed. A total of 2326, 260 and 1379 differential expressed genes (DEGs) were identified under the IAA, GR24 and IAA+GR24 treatments, respectively. Network analysis pointed out two enriched interaction clusters, including "ethylene biosynthesis" and "photosynthesis". Several ethylene biosynthesis and metabolism-related genes were up-regulated under both IAA and IAA+GR24 treatments, suggesting their involvement in the regulation of ethylene biosynthesis. Besides, auxin-SLs-triggered the expression of several CAB genes may lead to systemic increases in the induction of photosynthesis. Several auxin-activated metabolic pathways could be reduced by the GR24 treatment, indicated that the crosstalk between auxin and SLs may be involved in the metabolic regulation of tomato. Further analysis showed that SLs affect the responses of tomato shoots to auxin by inducing the expression of a series of auxin downstream genes. On the other hand, auxin regulated the biosynthesis of SLs by affecting the genes in the "Carotenoid biosynthesis" pathway. Our data will give us an opportunity to reveal the crosstalk between auxin and SLs in the shoots of tomato.

Identification and Expression Profiling of the Auxin Response Factors in Capsicum annuum L. under Abiotic Stress and Hormone Treatments.

Auxin response factors (ARFs) play important roles in regulating plant growth and development and response to environmental stress. An exhaustive analysis of the CaARF family was performed using the latest publicly available genome for pepper (Capsicum annuum L.). In total, 22 non-redundant CaARF gene family members in six classes were analyzed, including chromosome locations, gene structures, conserved motifs of proteins, phylogenetic relationships and Subcellular localization. Phylogenetic analysis of the ARFs from pepper (Capsicum annuum L.), tomato (Solanum lycopersicum L.), Arabidopsis and rice (Oryza sativa L.) revealed both similarity and divergence between the four ARF families, and aided in predicting biological functions of the CaARFs. Furthermore, expression profiling of CaARFs was obtained in various organs and tissues using quantitative real-time RT-PCR (qRT-PCR). Expression analysis of these genes was also conducted with various hormones and abiotic treatments using qRT-PCR. Most CaARF genes were regulated by exogenous hormone treatments at the transcriptional level, and many CaARF genes were altered by abiotic stress. Systematic analysis of CaARF genes is imperative to elucidate the roles of CaARF family members in mediating auxin signaling in the adaptation of pepper to a challenging environment.

Genome-wide identification and expression analysis of ClLAX, ClPIN and ClABCB genes families in Citrullus lanatus under various abiotic stresses and grafting.

BACKGROUND: Auxin plays an important role in regulating plant growth and development as well as in the response of plants to abiotic stresses. Auxin is transported by three kinds of major protein families, including the AUXIN RESISTANT 1/LIKE AUX1 (AUX/LAX) influx carriers, the PIN-FORMED (PIN) efflux carriers and the ATP binding cassette B/P-glycoprotein/Multidrug-resistance (ABCB/MDR/PGP) efflux/condition carriers. The biological function of several auxin transporter genes has been well characterized in Arabidopsis thaliana. However, their function in response to exogenous auxin and abiotic stresses in watermelon (Citrullus lanatus. L) remained unknown. RESULTS: Here, the latest updated watermelon genome was used to characterise the ClLAX, ClPIN and ClABCB family genes from watermelon. The genome-wide analysis of the ClLAX, ClPIN and ClABCB family genes, including chromosome localisation, gene structure, and phylogenic relationships, was carried out. Seven ClLAXs, 11 ClPINs and 15 ClABCBs were mapped on 10 watermelon chromosomes. The expression profiles of the ClLAX, ClPIN and ClABCB genes under exogenous indole-3-acetic acid and various abiotic stresses (salt, drought, and cold stresses) treatments were performed by quantitative real-time PCR (qRT-PCR). The transcriptional level of majority ClLAX, ClPIN and ClABCB genes were changed by abiotic stresses in both shoots and roots. We also analysed the expression levels of ClLAX, ClPIN and ClABCB genes in graft response. CONCLUSION: Analysis of the expression patterns of ClLAX, ClPIN and ClABCB genes under salt, drought, cold treatment and grafting response helps us to understand the possible roles of auxin transporter genes in watermelon adaptation to environmental stresses.

Identification of two transcription factors activating the expression of OsXIP in rice defence response.

BACKGROUND: Xylanase inhibitors have been confirmed to be involved in plant defence. OsXIP is a XIP-type rice xylanase inhibitor, yet its transcriptional regulation remains unknown. RESULTS: Herbivore infestation, wounding and methyl jasmonate (MeJA) treatment enhanced mRNA levels and protein levels of OsXIP. By analyzing different 5' deletion mutants of OsXIP promoter exposed to rice brown planthopper Nilaparvata lugens stress, a 562 bp region (-1451 - -889) was finally identified as the key sequence for the herbivores stress response. Using yeast one-hybrid screening, coupled with chromatin immunoprecipitation analysis, a basic helix-loop-helix protein (OsbHLH59) and an APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor OsERF71 directly binding to the 562 bp key sequence to activate the expression of OsXIP were identified, which is further supported by transient expression assay. Moreover, transcriptional analysis revealed that mechanical wounding and treatment with MeJA resulted in an obvious increase in transcript levels of OsbHLH59 and OsERF71 in root and shoot tissues. CONCLUSIONS: Our data shows that two proteins as direct transcriptional activators of OsXIP responding to stress were identified. These results reveal a coordinated regulatory mechanism of OsXIP, which may probably be involved in defence responses via a JA-mediated signaling pathway.