Update on functional analysis of long non-coding RNAs in common crops.

With the rapid advances in next-generation sequencing technology, numerous non-protein-coding transcripts have been identified, including long noncoding RNAs (lncRNAs), which are functional RNAs comprising more than 200 nucleotides. Although lncRNA-mediated regulatory processes have been extensively investigated in animals, there has been considerably less research on plant lncRNAs. Nevertheless, multiple studies on major crops showed lncRNAs are involved in crucial processes, including growth and development, reproduction, and stress responses. This review summarizes the progress in the research on lncRNA roles in several major crops, presents key strategies for exploring lncRNAs in crops, and discusses current challenges and future prospects. The insights provided in this review will enhance our comprehension of lncRNA functions in crops, with potential implications for improving crop genetics and breeding.

Expression Patterns and Molecular Mechanisms Regulating Drought Tolerance of Soybean [Glycine max (L.) Merr.] Conferred by Transcription Factor Gene GmNAC19.

NAC transcription factors are commonly involved in the plant response to drought stress. A transcriptome analysis of root samples of the soybean variety 'Jiyu47' under drought stress revealed the evidently up-regulated expression of GmNAC19, consistent with the expression pattern revealed by quantitative real-time PCR analysis. The overexpression of GmNAC19 enhanced drought tolerance in Saccharomyces cerevisiae INVSc1. The seed germination percentage and root growth of transgenic Arabidopsis thaliana were improved in comparison with those of the wild type, while the transgenic soybean composite line showed improved chlorophyll content. The altered contents of physiological and biochemical indices (i.e., soluble protein, soluble sugar, proline, and malondialdehyde) related to drought stress and the activities of three antioxidant enzymes (i.e., superoxide dismutase, peroxidase, and catalase) revealed enhanced drought tolerance in both transgenic Arabidopsis and soybean. The expressions of three genes (i.e., P5CS, OAT, and P5CR) involved in proline synthesis were decreased in the transgenic soybean hairy roots, while the expression of ProDH involved in the breakdown of proline was increased. This study revealed the molecular mechanisms underlying drought tolerance enhanced by GmNAC19 via regulation of the contents of soluble protein and soluble sugar and the activities of antioxidant enzymes, providing a candidate gene for the molecular breeding of drought-tolerant crop plants.

Saline-Alkali Soil Property Improved by the Synergistic Effects of Priestia aryabhattai JL-5, Staphylococcus pseudoxylosus XW-4, Leymus chinensis and Soil Microbiota.

Two saline-alkali-tolerant bacterial strains, Priestia aryabhattai JL-5 and Staphylococcus pseudoxylosus XW-4, were isolated, with high capabilities of hydrolyzing phosphate and producing cellulase, respectively. The molecular mechanisms regulating the saline-alkali tolerance in the strain JL-5 were further investigated using transcriptome analysis. The contents of lactic acid and proline and the enzymatic activity of glutamine synthetase in the strain JL-5 were significantly increased. The properties of saline-alkali soils were significantly improved by the enhanced growth of the indicator plant Leymus chinensis under the combined applications of the strains JL-5 and XW-4 mixed with corn straw. The contents of catalase, peroxidase, superoxide dismutase and proline of L. chinensis were significantly increased, and the content of malondialdehyde was significantly decreased in the combined treatment of both bacterial strains. The contents of available nitrogen, phosphorus and potassium and organic matters in the soil treated with both strains were significantly increased, as well as the diversity and abundance of the soil microbiota. Our study evidently demonstrated the synergistic effects of the strains JL-5 and XW-4, indicator plants and the local microbiota in terms of improving the saline-alkali soil properties, providing strong experimental evidence to support the commercial development of the combined application of both strains to improve the properties of saline-alkali soils.

Critical Role of Monooxygenase in Biodegradation of 2,4,6-Trinitrotoluene by Buttiauxella sp. S19-1.

2,4,6-Trinitrotoluene (TNT) is an aromatic pollutant that is difficult to be degraded in the natural environment. The screening of efficient degrading bacteria for bioremediation of TNT has received much attention from scholars. In this paper, transcriptome analysis of the efficient degrading bacterium Buttiauxella sp. S19-1 revealed that the monooxygenase gene (BuMO) was significantly up-regulated during TNT degradation. S-ΔMO (absence of BuMO gene in S19-1 mutant) degraded TNT 1.66-fold less efficiently than strain S19-1 (from 71.2% to 42.9%), and E-MO mutant (Escherichia coli BuMO-expressing strain) increased the efficiency of TNT degradation 1.33-fold (from 52.1% to 69.5%) for 9 h at 180 rpm at 27 °C in LB medium with 1.4 µg·mL-1 TNT. We predicted the structure of BuMO and purified recombinant BuMO (rBuMO). Its specific activity was 1.81 µmol·min-1·mg-1 protein at pH 7.5 and 35 °C. The results of gas chromatography mass spectrometry (GC-MS) analysis indicated that 4-amino-2,6-dinitrotoluene (ADNT) is a metabolite of TNT biodegradation. We speculate that MO is involved in catalysis in the bacterial degradation pathway of TNT in TNT-polluted environment.

Potential Effects of Metal Oxides on Agricultural Production of Rice: A Mini Review.

The extensive usage of metal oxide nanoparticles has aided in the spread and accumulation of these nanoparticles in the environment, potentially endangering both human health and the agroecological system. This research describes in detail the hazardous and advantageous impacts of common metal oxide nanomaterials, such as iron oxide, copper oxide, and zinc oxide, on the life cycle of rice. In-depth analyses are conducted on the transport patterns of nanoparticles in rice, the plant's reaction to stress, the reduction of heavy metal stress, and the improvement of rice quality by metal oxide nanoparticles, all of which are of significant interest in this subject. It is emphasized that from the perspective of advancing the field of nanoagriculture, the next stage of research should focus more on the molecular mechanisms of the effects of metal oxide nanoparticles on rice and the effects of combined use with other biological media. The limitations of the lack of existing studies on the effects of metal oxide nanomaterials on the entire life cycle of rice have been clearly pointed out.

Molecular Characterization and Drought Resistance of GmNAC3 Transcription Factor in Glycine max (L.) Merr.

Soybean transcription factor GmNAC plays important roles in plant resistance to environmental stresses. In this study, GmNAC3 was cloned in the drought tolerant soybean variety "Jiyu47", with the molecular properties of GmNAC3 characterized to establish its candidacy as a NAC transcription factor. The yeast self-activation experiments revealed the transcriptional activation activity of GmNAC3, which was localized in the nucleus by the subcellular localization analysis. The highest expression of GmNAC3 was detected in roots in the podding stage of soybean, and in roots of soybean seedlings treated with 20% PEG6000 for 12 h, which was 16 times higher compared with the control. In the transgenic soybean hairy roots obtained by the Agrobacterium-mediated method treated with 20% PEG6000 for 12 h, the activities of superoxide dismutase, peroxidase, and catalase and the content of proline were increased, the malondialdehyde content was decreased, and the expressions of stress resistance-related genes (i.e., APX2, LEA14, 6PGDH, and P5CS) were up-regulated. These expression patterns were confirmed by transgenic Arabidopsis thaliana with the overexpression of GmNAC3. This study provided strong scientific evidence to support further investigation of the regulatory function of GmNAC3 in plant drought resistance and the molecular mechanisms regulating the plant response to environmental stresses.

Proteomic Investigation of Molecular Mechanisms in Response to PEG-Induced Drought Stress in Soybean Roots.

Roots are generally the critical drought sensors, but little is known about their molecular response to drought stress. We used the drought-tolerant soybean variety 'Jiyu 47' to investigate the differentially expressed proteins (DEPs) in soybean roots during the seedling stage based on the tandem mass tag (TMT) proteomics analysis. Various expression patterns were observed in a total of six physiological parameters. A total of 468 DEPs (144 up-regulated and 324 down-regulated) among a total of 8687 proteins were identified in response to drought stress in 24 h. The expression of DEPs was further validated based on quantitative real-time PCR of a total of five genes (i.e., GmGSH, GmGST1, GmGST2 k GmCAT, and Gm6PGD) involved in the glutathione biosynthesis. Results of enrichment analyses revealed a coordinated expression pattern of proteins involved in various cellular metabolisms responding to drought stress in soybean roots. Our results showed that drought stress caused significant alterations in the expression of proteins involved in several metabolic pathways in soybean roots, including carbohydrate metabolism, metabolism of the osmotic regulation substances, and antioxidant defense system (i.e., the glutathione metabolism). Increased production of reduced glutathione (GSH) enhanced the prevention of the damage caused by reactive oxygen species and the tolerance of the abiotic stress. The glutathione metabolism played a key role in modifying the antioxidant defense system in response to drought stress in soybean roots. Our proteomic study suggested that the soybean plants responded to drought stress by coordinating their protein expression during the vegetative stage, providing novel insights into the molecular mechanisms regulating the response to abiotic stress in plants.

Genome-Wide Analysis of the DUF4228 Family in Soybean and Functional Identification of GmDUF4228 -70 in Response to Drought and Salt Stresses.

Domain of unknown function 4228 (DUF4228) proteins are a class of proteins widely found in plants, playing an important role in response to abiotic stresses. However, studies on the DUF4228 family in soybean (Glycine max L.) are sparse. In this study, we identified a total of 81 DUF4228 genes in soybean genome, named systematically based on their chromosome distributions. Results showed that these genes were unevenly distributed on the 20 chromosomes of soybean. The predicted soybean DUF4228 proteins were identified in three groups (Groups I-III) based on a maximum likelihood phylogenetic tree. Genetic structure analysis showed that most of the GmDUF4228 genes contained no introns. Expression profiling showed that GmDUF4228 genes were widely expressed in different organs and tissues in soybean. RNA-seq data were used to characterize the expression profiles of GmDUF4228 genes under the treatments of drought and salt stresses, with nine genes showing significant up-regulation under both drought and salt stress further functionally verified by promoter (cis-acting elements) analysis and quantitative real-time PCR (qRT-PCR). Due to its upregulation under drought and salt stresses based on both RNA-seq and qRT-PCR analyses, GmDUF4228-70 was selected for further functional analysis in transgenic plants. Under drought stress, the degree of leaf curling and wilting of the GmDUF4228-70-overexpressing (GmDUF4228-70-OE) line was lower than that of the empty vector (EV) line. GmDUF4228-70-OE lines also showed increased proline content, relative water content (RWC), and chlorophyll content, and decreased contents of malondialdehyde (MDA), H2O2, and O2-. Under salt stress, the changes in phenotypic and physiological indicators of transgenic plants were the same as those under drought stress. In addition, overexpression of the GmDUF4228-70 gene promoted the expression of marker genes under both drought and salt stresses. Taken together, the results indicated that GmDUF4228 genes play important roles in response to abiotic stresses in soybean.

Genomic Analysis of Stress Associated Proteins in Soybean and the Role of GmSAP16 in Abiotic Stress Responses in Arabidopsis and Soybean.

Stress associated proteins (SAPs) containing A20/AN1 zinc finger domains have emerged as novel regulators of stress responses. In this study, 27 SAP genes were identified in soybean. The phylogenetic relationships, exon-intron structure, domain structure, chromosomal localization, putative cis-acting elements, and expression patterns of SAPs in various tissues under abiotic stresses were analyzed. Among the soybean SAP genes, GmSAP16 was significantly induced by water deficit stress, salt, and abscisic acid (ABA) and selected for further analysis. GmSAP16 was located in the nucleus and cytoplasm. The overexpression of GmSAP16 in Arabidopsis improved drought and salt tolerance at different developmental stages and increased ABA sensitivity, as indicated by delayed seed germination and stomatal closure. The GmSAP16 transgenic Arabidopsis plants had a higher proline content and a lower water loss rate and malondialdehyde (MDA) content than wild type (WT) plants in response to stresses. The overexpression of GmSAP16 in soybean hairy roots enhanced drought and salt tolerance of soybean seedlings, with higher proline and chlorophyll contents and a lower MDA content than WT. RNA inference (RNAi) of GmSAP16 increased stress sensitivity. Stress-related genes, including GmDREB1B;1, GmNCED3, GmRD22, GmDREB2, GmNHX1, and GmSOS1, showed significant expression alterations in GmSAP16-overexpressing and RNAi plants under stress treatments. These results indicate that soybean SAP genes play important roles in abiotic stress responses.

Genome-Wide Characterization and Expression Analysis of Soybean TGA Transcription Factors Identified a Novel TGA Gene Involved in Drought and Salt Tolerance.

The TGA transcription factors, a subfamily of bZIP group D, play crucial roles in various biological processes, including the regulation of growth and development as well as responses to pathogens and abiotic stress. In this study, 27 TGA genes were identified in the soybean genome. The expression patterns of GmTGA genes showed that several GmTGA genes are differentially expressed under drought and salt stress conditions. Among them, GmTGA17 was strongly induced by both stress, which were verificated by the promoter-GUS fusion assay. GmTGA17 encodes a nuclear-localized protein with transcriptional activation activity. Heterologous and homologous overexpression of GmTGA17 enhanced tolerance to drought and salt stress in both transgeinc Arabidopsis plants and soybean hairy roots. However, RNAi hairy roots silenced for GmTGA17 exhibited an increased sensitivity to drought and salt stress. In response to drought or salt stress, transgenic Arabidopsis plants had an increased chlorophyll and proline contents, a higher ABA content, a decreased MDA content, a reduced water loss rate, and an altered expression of ABA- responsive marker genes compared with WT plants. In addition, transgenic Arabidopsis plants were more sensitive to ABA in stomatal closure. Similarly, measurement of physiological parameters showed an increase in chlorophyll and proline contents, with a decrease in MDA content in soybean seedlings with overexpression hairy roots after drought and salt stress treatments. The opposite results for each measurement were observed in RNAi lines. This study provides new insights for functional analysis of soybean TGA transcription factors in abiotic stress.

ZmCOL3, a CCT gene represses flowering in maize by interfering with the circadian clock and activating expression of ZmCCT.

Flowering time is a trait vital to the adaptation of flowering plants to different environments. Here, we report that CCT domain genes play an important role in flowering in maize (Zea mays L.). Among the 53 CCT family genes we identified in maize, 28 were located in flowering time quantitative trait locus regions and 15 were significantly associated with flowering time, based on candidate-gene association mapping analysis. Furthermore, a CCT gene named ZmCOL3 was shown to be a repressor of flowering. Overexpressing ZmCOL3 delayed flowering time by approximately 4 d, in either long-day or short-day conditions. The absence of one cytosine in the ZmCOL3 3'UTR and the presence of a 551 bp fragment in the promoter region are likely the causal polymorphisms contributing to the maize adaptation from tropical to temperate regions. We propose a modified model of the maize photoperiod pathway, wherein ZmCOL3 acts as an inhibitor of flowering either by transactivating transcription of ZmCCT, one of the key genes regulating maize flowering, or by interfering with the circadian clock.

Genome-wide analysis of the YABBY family in soybean and functional identification of GmYABBY10 involvement in high salt and drought stresses.

YABBY family is a plant specific transcription factor family, with the typical N-terminal C2C2 type zinc finger domain and the C-terminal YABBY conservative structure domain, which plays important biological roles in plant growth, development and morphogenesis. In this study, a total of 17 YABBY genes were identified in the soybean genome. The results of this research showed that 17 soybean YABBY genes were located on 11 chromosomes. Analysis of putative cis-acting elements showed that soybean YABBY genes contained lots of MYB and MYC elements. Quantitative Real-time PCR (qRT-PCR) showed that the expressions of GmYABBY3, GmYABBY10 and GmYABBY16 were more highly sensitive in drought, NaCl and ABA stresses. And the transient expression in Arabidopsis protoplasts showed that GmYABBY3 protein distributed uniformly the whole cells, while GmYABBY10 protein was mainly localized in the membranes and cytoplasm and GmYABBY16 protein was localized the nucleus and membranes. To further identify the function of GmYABBY10, we obtained the transgenic Arabidopsis overexpression GmYABBY10. Based on germination and seedling root arrays in transgenic Arabidopsis, we found that the rates of wild type seeds was a litter higher than that of GmYABBY10 transgenic seeds under both PEG and NaCl treatment. While the root length and root surface of wild type seedlings were bigger than those of GmYABBY10 transgenic seedlings. When seedlings were grown in soil, the survival rates of wild type were higher than those of transgenic plants under both PEG and NaCl treatment, which indicated that GmYABBY10 may be a negatively regulator in plant resistances to drought and salt stresses. This study provided valuable information regarding the classification and functions of YABBY genes in soybean.

Tissue-Specific Regulation of Gma-miR396 Family on Coordinating Development and Low Water Availability Responses.

Previously, it was reported that miR396s interact with growth-regulating factors (GRFs) to modulate plant growth, development, and stress resistance. In soybean, 11 gma-miR396 precursors (Pre-miR396a-k) were found, and 24 GmGRFs were predicted as targets of seven mature gma-miR396s (gma-miR396a/b/c/e/h/i/k). To explore the roles of the miR396-GRF module in low water availability response of soybean, we analyzed the expression of Pre-miR396a-k, and found that Pre-miR396a/i/bdgk/e/h were up-regulated in leaves and down-regulated in roots; on the contrary, GmGRF5/6/7/8/15/17/21 were down-regulated in leaves and GmGRF1/2/17/18/19/20/21/22/23/24 were up-regulated in roots of low water potential stressed soybean. Any one of gma-miR396a/b/c/e/h/i/k was able to interact with 20 GmGRFs (GmGRF1/2/6-11/13-24), confirming that this module represents a multi-to-multi network interaction. We generated Arabidopsis plants over-expressing each of the 11 gma-miR396 precursors (Pre-miR396a-k), and seven of them (miR396a/b/c/e/h/i/k-OE transgenic Arabidopsis) showed altered development. The low water availability of miR396a/b/c/e/h/i/k-OE was enhanced in leaves but reduced in seeds and roots. Contrary to previous reports, miR396a/b/c/i-OE seedlings showed lower survival rate than WT when recovering after rewatering under soil drying. In general, we believe our findings are valuable to understand the role of gma-miR396 family in coordinating development and low water availability responses, and can provide potential strategies and directions for soybean breeding programs to improve seed yield and plant drought tolerance.

De Novo Sequencing and Analysis of the Safflower Transcriptome to Discover Putative Genes Associated with Safflor Yellow in Carthamus tinctorius L.

Safflower (Carthamus tinctorius L.), an important traditional Chinese medicine, is cultured widely for its pharmacological effects, but little is known regarding the genes related to the metabolic regulation of the safflower's yellow pigment. To investigate genes related to safflor yellow biosynthesis, 454 pyrosequencing of flower RNA at different developmental stages was performed, generating large databases.In this study, we analyzed 454 sequencing data from different flowering stages in safflower. In total, 1,151,324 raw reads and 1,140,594 clean reads were produced, which were assembled into 51,591 unigenes with an average length of 679 bp and a maximum length of 5109 bp. Among the unigenes, 40,139 were in the early group, 39,768 were obtained from the full group and 28,316 were detected in both samples. With the threshold of "log2 ratio ≥ 1", there were 34,464 differentially expressed genes, of which 18,043 were up-regulated and 16,421 were down-regulated in the early flower library. Based on the annotations of the unigenes, 281 pathways were predicted. We selected 12 putative genes and analyzed their expression levels using quantitative real time-PCR. The results were consistent with the 454 sequencing results. In addition, the expression of chalcone synthase, chalcone isomerase and anthocyanidin synthase, which are involved in safflor yellow biosynthesis and safflower yellow pigment (SYP) content, were analyzed in different flowering periods, indicating that their expression levels were related to SYP synthesis. Moreover, to further confirm the results of the 454 pyrosequencing, full-length cDNA of chalcone isomerase (CHI) and anthocyanidin synthase (ANS) were cloned from safflower petal by RACE (Rapid-amplification of cDNA ends) method according to fragment of the transcriptome.

The soybean GmDi19-5 interacts with GmLEA3.1 and increases sensitivity of transgenic plants to abiotic stresses.

Drought-induced (Di19) proteins played important roles in plant growth, development, and abiotic stress responses. In the present study, a total of seven Di19 genes were identified in soybean. Each soybean Di19 gene showed specific responses to salt, drought, oxidative, and ABA stresses based on expression profiles. With a relatively higher transcript level among Di19 members under four stress treatments, GmDi19-5 was selected for detailed analysis. Inhibitor assays revealed that ABA inhibitor (Fluridone) or H2O2 inhibitor (DMTU) was involved in the drought- or salt-induced transcription of GmDi19-5. The GUS activity driven by the GmDi19-5 promoter was induced by salt, PEG, ABA, and MV treatments and tended to be accumulated in the vascular bundles and young leaves. A subcellular localization assay showed that GmDi19-5 protein localized in the nucleus. Further investigation showed that GmDi19-5 protein was involved in the interaction with GmLEA3.1. Overexpression of GmDi19-5 increased sensitivity of transgenic Arabidopsis plants to salt, drought, oxidative, and ABA stresses and regulated expression of several ABA/stress-associated genes. This present investigation showed that GmDi19-5 functioned as a negative factor under abiotic stresses and was involved in ABA and SOS signaling pathway by altering transcription of stress-associated genes.

Discovery and analysis of microRNAs in Leymus chinensis under saline-alkali and drought stress using high-throughput sequencing.

Leymus chinensis (Trin.) Tzvel. is a perennial rhizome grass of the Poaceae (also called Gramineae) family, which adapts well to drought, saline and alkaline conditions. However, little is known about the stress tolerance of L. chinensis at the molecular level. microRNAs (miRNAs) are known to play critical roles in nutrient homeostasis, developmental processes, pathogen responses, and abiotic stress in plants. In this study, we used Solexa sequencing technology to generate high-quality small RNA data from three L. chinensis groups: a control group, a saline-alkaline stress group (100 mM NaCl and 200 mM NaHCO3), and a drought stress group (20% polyethylene glycol 2000). From these data we identified 132 known miRNAs and 16 novel miRNAs candidates. For these miRNAs we also identified target genes that encode a broad range of proteins that may be correlated with abiotic stress regulation. This is the first study to demonstrate differentially expressed miRNAs in L. chinensis under saline-alkali and drought stress. These findings may help explain the saline-alkaline and drought stress responses in L. chinensis.

[Monthly dynamics and distribution of major cations in Iris lactea].

The study on the monthly absorption, transportation, and distribution of Na+, K+, Ca2+ and Mg2+ in Iris lactea under saline-alkali field conditions showed that the contents of test cations in I. lactea varied with months. After June, the cations contents in plant increased with growth. Root Ca2+ and Na+ contents were the highest in July, being 2.30% and 0.51%, respectively, while root K+ and Mg2+ contents were the highest in September (0.27%) and October (0.28%), respectively. Leaf Na+ content was the highest in July (0.57%), while leaf K+, Ca2+ and Mg2+ contents were the highest in August, being 1.30%, 2.69% and 0.47%, respectively. In July and August, the selective absorption (SA) of K+ was higher than that of Na+, while the selective transport (ST) was in adverse. The cations contents in I. lactea were significantly higher than those in soil, suggesting that I. lactea had high accumulation capacity to these cations. The cations were mainly accumulated in the 0-30 cm aboveground part and 0-40 cm underground part of I. lactea, and the average contents of Na+, K+, Ca2+ and Mg2+ in aboveground part were 9.11, 4.07, 0.98 and 2.27 times of those in underground part, respectively.