Investigation of <i>GhFAT</i> Genes Related to Seed Oil Content and Fatty Acid Composition in <i>Gossypium hirsutum</i> L.

Fatty Acyl-ACP thioesterase (FAT) is a key enzyme controlling oil biosynthesis in plant seeds. FATs can be divided into two subfamilies, FATA and FATB according to their amino acid sequences and substrate specificity. The Upland cotton genome contains 20 GhFAT genes, amongst which 6 genes were of the GhFATA subfamily and 14 of the GhFATB subfamily. The 20 GhFAT genes are unevenly distributed on 14 chromosomes. The GhFATA genes have 5 or 7 exons and the GhFATB genes have 6 or 7 exons. All GhFAT proteins have the conserved Acyl-ACP_TE domain and PLN02370 super family, the typical characteristics of plant thioesterases. Analyses of the expression level of GhFATs and the compositions of fatty acid in 5–60 days-post-anthesis seeds showed that the ratio of saturated fatty acids to unsaturated fatty acids was consistent with the expression profile of GhFATB12, GhFATB3, and GhFATB10; the ratio of monounsaturated fatty acid to polyunsaturated fatty acids was consistent with the expression profile of GhFATA3. The oil contents of mature cottonseeds were positively correlated with the contents of palmitic acid and linolenic acid as well as seed vigor. These results provide essential information for further exploring the role(s) of the specific GhFATs in determining oil biosynthesis and cottonseed compositions.     

Genome-Wide Identification and Expression Profiling of the Shaker K⁺ Channel and HAK/KUP/KT Transporter Gene Families in Grape (<i>Vitis vinifera</i> L.)

Potassium (K⁺) is an essential macronutrient for plants to maintain normal growth and development. Shaker-like K⁺ channels and HAK/KUP/KT transporters are critical components in the K⁺ acquisition and translocation. In this study, we identified 9 Shaker-like K⁺ channel (VvK) and 18 HAK/KUP/KT transporter (VvKUP) genes in grape, which were renamed according to their distributions in the genome and relative linear orders among the distinct chromosomes. Similar structure organizations were found within each group according to the exon/intron structure and protein motif analysis. Chromosomal distribution analysis showed that 9 VvK genes and 18 VvKUP genes were unevenly distributed on 7 or 10 putative grape chromosomes. Three pairs of tandem duplicated genes and one pair of segmental duplicated genes were observed in the expansion of the grape VvKUP genes. Gene expression omnibus (GEO) data analysis showed that VvK and VvKUP genes were expressed differentially in distinct tissues. Various cis-acting regulatory elements pertinent to phytohormone responses and abiotic stresses, including K⁺ deficiency response and drought stress, were detected in the promoter region of VvK and VvKUP genes. This study provides valuable information for further functional studies of VvK and VvKUP genes, and lays a foundation to explore K⁺ uptake and utilization in fruit trees.     

Overexpression of <i>IbSINA5</i> Increases Cold Tolerance through a CBF SINA-COR Mediated Module in Sweet Potato

Seven in absentia (SINA) family proteins play a central role in plant growth, development and resistance to abiotic stress. However, their biological function in plant response to cold stress is still largely unknown. In this work, a seven in absentia gene IbSINA5 was isolated from sweet potato. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses demonstrated that IbSINA5 was ubiquitously expressed in various tissues and organs of sweet potato, with a predominant expression in fibrous roots, and was remarkably induced by cold, drought and salt stresses. Subcellular localization assays revealed that IbSINA5-GFP fusion protein was mainly localized in cytoplasm and nucleus. Overexpression of IbSINA5 in sweet potato led to dramatically improved resistance to cold stress in transgenic plants, which was associated with the up-regulated expression of IbCOR (cold-regulated) genes, increased proline production, and decreased malondialdehyde (MDA) and H₂O₂ accumulation in the leaves of transgenic plants. Furthermore, transient expression of IbCBF3, a C-repeat binding factor (CBF) gene, in the leaf protoplasts of wild type sweet potato plants up-regulated the expression of both IbSINA5 and IbCOR genes. Our results suggest that IbSINA5 could function as a positive regulator in the cold signaling pathway through a CBF-SINA-COR mediated module in sweet potato, and have a great potential to be used as a candidate gene for the future breeding of new plant species with improved cold resistance.     

<i>Aspergillus tubingensis</i> Causes Leaf Spot of Cotton (<i>Gossypium hirsutum</i> L.) in Pakistan

Cotton (Gossypium hirsutum L.) is a key fiber crop of great commercial importance. Numerous phytopathogens decimate crop production by causing various diseases. During July-August 2018, leaf spot symptoms were recurrently observed on cotton leaves in Rahim Yar Khan, Pakistan and adjacent areas. Infected leaf samples were collected and plated on potato dextrose agar (PDA) media. Causal agent of cotton leaf spot was isolated, characterized and identified as Aspergillus tubingensis based on morphological and microscopic observations. Conclusive identification of pathogen was done on the comparative molecular analysis of CaM and β-tubulin gene sequences. BLAST analysis of both sequenced genes showed 99% similarity with A. tubingensis. Koch’s postulates were followed to confirm the pathogenicity of the isolated fungus. Healthy plants were inoculated with fungus and similar disease symptoms were observed. Fungus was re-isolated and identified to be identical to the inoculated fungus. To our knowledge, this is the first report describing the involvement of A. tubingensis in causing leaf spot disease of cotton in Pakistan and around the world.     

Differential Responses of <i>NHX1</i> and <i>SOS1</i> Gene Expressions to Salinity in two <i>Miscanthus sinensis</i> Anderss. Accessions with Different Salt Tolerance

The lignocellulosic crop Miscanthus spp. has been identified as a good candidate for biomass production. The responses of Miscanthus sinensis Anderss. to salinity were studied to satisfy the needs for high yields in marginal areas and to avoid competition with food production. The results indicated that the relative advantages of the tolerant accession over the sensitive one under saline conditions were associated with restricted Na⁺ accumulation in shoots. Seedlings of two accessions (salt-tolerant ‘JM0119’ and salt-sensitive ‘JM0099’) were subjected to 0 (control), 100, 200, and 300 mM NaCl stress to better understand the salt-induced biochemical responses of genes involved in Na⁺ accumulation in M. sinensis. The adaptation responses of genes encoding for Na⁺ /H⁺ antiporters, NHX1 and SOS1 to NaCl stress were examined in JM0119 and JM0099.The cDNA sequences of genes examined were highly conserved among the relatives of M. sinensis based on the sequencing on approximate 600 bp-long cDNA fragments obtained from degenerate PCR. These salt-induced variations of gene expression investigated by quantitative real-time PCR provided evidences for insights of the molecular mechanisms of salt tolerance in M. sinensis. The expression of NHX1 was up-regulated by salt stress in JM0119 shoot and root tissues. However, it was hardly affected in JM0099 shoot tissue except for a significant increase at the 100 mM salt treatment, and it was salt-suppressed in the JM0099 root tissue. In the root tissue, the expression of SOS1 was induced by the high salt treatment in JM0119 but repressed by all salt treatments in JM0099. Thus, the remarkably higher expression of NHX1 and SOS1 were associated with the resistance to Na⁺ toxicity by regulation of the Na⁺ influx, efflux, and sequestration under different salt conditions.     

Genome-Wide Analysis of the <i>KANADI</i> Gene Family and Its Expression Patterns under Different Nitrogen Concentrations Treatments in <i>Populus trichocarpa</i>

KANADI (KAN) is a plant-specific gene that controlled the polarity development of lateral organs. It mainly acted on the abaxial characteristics of plants to make the lateral organs asymmetrical. However, it had been less identified in woody plants. In this study, the members of the KAN gene family in Populus trichocarpa were identified and analyzed using the bioinformatics method. The results showed that a total of 8 KAN family members were screened out, and each member contained the unique GARP domain and conserved region of the family proteins. Phylogenetic analysis and their gene structures revealed that all KAN genes from P. trichocarpa, Arabidopsis thaliana, and Nicotiana benthamiana could be divided into four subgroups, while the eight genes in P. trichocarpa were classified into three subgroups, respectively. The analysis of tissue-specific expression indicated that PtKAN1 was highly expressed in young leaves, PtKAN6 was highly expressed in young leaves and mature leaves, PtKAN2, PtKAN5, and PtKAN7 were highly expressed in nodes and internodes, PtKAN8 was highly expressed in roots, and PtKAN3 and PtKAN4 showed low expression levels in all tissues. Among them, PtKAN2 and PtKAN6, and PtKAN4 and PtKAN5 might have functional redundancy. Under high nitrogen concentrations, PtKAN2 and PtKAN8 were highly expressed in mature stems and leaves, respectively, while PtKAN4, PtKAN5, and PtKAN7 were highly expressed in roots. This study laid a theoretical foundation for further study of the KAN gene-mediated nitrogen effect on root development.     

Genome-Wide Identification of Candidate Genes Associated with <i>β</i>-glucan Traits in a Hulled and Hulless Barley (<i>Hordeum vulgare</i> L.) Population

Barley grain is a valuable source of β-glucan, which is an important component of dietary fiber with significant human health benefits. Although the genetic basis of β-glucan biosynthesis has been widely studied, a genome-wide association study (GWAS) is still required for a scan of the candidate genes related to the complex quantitative trait based on the high-quality barley reference genome. In this study, a GWAS was conducted using a population composed of 87 barley landraces (39 hulled and 48 hulless, β-glucan from 2.07% to 6.56%) with 191,098 nucleotide polymorphisms (SNPs) markers to cover the chromosomes with the highest density. The population was divided into four sub-populations (POP1~POP4), and the β-glucan content in POP2 was significantly higher than that in other groups, in which most of the hulless barley landraces are from Qinghai-Tibet Plateau in China. Among seven SNP markers identified by GWAS, two (SNP2 and SNP3) of them showed positive correlation to β-glucan trait and the remaining five (SNP1, SNP4, SNP5, SNP6 and SNP7) showed the negative relationship. Two candidate genes linked to SNP7, HORVU7Hr1G000320 and HORVU7Hr1G000040, belong to the nucleotide triphosphate hydrolase superfamily which is probable to affect the activities of β-glucan synthase. Another candidate gene associated with SNP1, HORVU1Hr1G000010, is possibly involved in sugar response. In conclusion, our results provide new insights into the genetic basis of β-glucan accumulation in barley grains, and the discovery of new SNP markers distributed in each chromosome and the associated candidate genes will be valuable for the breeding of functional barley varieties with high β-glucan.     

Identification of a Novel <i>OsCYP2</i> Allele that Was Involved in Rice Response to Low Temperature Stress

Cyclophilin (CYP) plays an important role in plant response to stress, and OsCYP2, one gene of cyclophlilin family, is involved in auxin signal transduction and stress signaling in rice. However, the mechanism that OsCYP2 is involved in rice response to low temperature is still unclear. We identified a new OsCYP2 allelic mutant, lrl3, with fewer lateral roots, and the differences in shoot height, primary root length and adventitious root length increased with the growth process compared to the wild-type plant. Auxin signaling pathway was also affected and became insensitive to gravity. The transgenic rice plants with over-expression of OsCYP2 were more tolerant to low temperature than the wild-type plants, suggesting that OsCYP2 was involved in the low temperature response in rice. In addition, OsCYP2 negatively regulated the expression of OsTPS38, a terpene synthase gene, and was dependent on the OsCDPK7-mediated pathway in response to low temperature stress. OsTPS38- overexpressed transgenic line ox-2 was more sensitive to low temperature. Therefore, OsCYP2 may negatively regulate OsTPS38 through an OsCDPK7-dependent pathway to mediate the response to low temperature in rice. These results provide a new basis for auxin signaling genes to regulate rice response to low temperature stress.     

Overexpression of Wheat <i>TaELF3-1BL</i> Delays Flowering in Arabidopsis

EARLY FLOWERING 3 (ELF3), a light zeitnehmer (time-taker) gene, regulates circadian rhythm and photoperiodic flowering in Arabidopsis, rice, and barley. The three orthologs of ELF3 (TaELF3-1AL, TaELF3-1BL, and TaELF3-1DL) have been identified in wheat too, and one gene, TaELF3-1DL, has been associated with heading date. However, the basic characteristics of these three genes and the roles of the other two genes, TaELF3-1BL and, TaELF3-1AL, remain unknown. Therefore, the present study obtained the coding sequences of the three orthologs (TaELF3-1AL, TaELF3-1BL, and TaELF3-1DL) of ELF3 from bread wheat and characterized them and investigated the role of TaELF3-1BL in Arabidopsis. Protein sequence comparison revealed similarities among the three TaELF3 genes of wheat; however, they were different from the Arabidopsis ELF3. Real-time quantitative PCR revealed TaELF3 expression in all wheat tissues tested, with the highest expression in young spikes; the three genes showed rhythmic expression patterns also. Furthermore, the overexpression of the TaELF3-1BL gene in Arabidopsis delayed flowering, indicating their importance in flowering. Subsequent overexpression of TaELF3-1BL in the Arabidopsis ELF3 nonfunctional mutant (elf3 mutant) eliminated its early flowering phenotype, and slightly delayed flowering. The wild-type Arabidopsis overexpressing TaELF3-1BL demonstrated reduced expression levels of flowering-related genes, such as CONSTANS (AtCO), FLOWERING LOCUS T (AtFT), and GIGANTEA (AtGI). Thus, the study characterized the three TaELF3 genes and associated TaELF3-1BL with flowering in Arabidopsis, suggesting a role in regulating flowering in wheat too. These findings provide a basis for further research on TaELF3 functions in wheat.     

Characterization and Expression of Ammonium Transporter in Peach (<i>Prunus persica</i>) and Regulation Analysis in Response to External Ammonium Supply

As the preferred nitrogen (N) source, ammonium (NH₄⁺ ) contributes to plant growth and development and fruit quality. In plants, NH₄⁺ uptake is facilitated by a family of NH₄⁺ transporters (AMT). However, the molecular mechanisms and functional characteristics of the AMT genes in peach have not been mentioned yet. In this present study, excess NH₄⁺ stress severely hindered shoot growth and root elongation, accompanied with reduced mineral accumulation, decreased leaf chlorophyll concentration, and stunned photosynthetic performance. In addition, we identified 14 putative AMT genes in peach (PpeAMT). Expression analysis showed that PpeAMT genes were differently expressed in peach leaves, stems and roots, and were distinctly regulated by external NH₄⁺ supplies. Putative cis-elements involved in abiotic stress adaption, Ca²⁺ response, light and circadian rhythms regulation, and seed development were observed in the promoters of the PpeAMT family genes. Phosphorylation analysis of residues within the C-terminal of PpeAMT proteins revealed many conserved phosphorylation residues in both the AMT1 and AMT2 subfamily members, which could potentially play roles in controlling the NH₄⁺ transport activities. This study provides gene resources to study the biological function of AMT proteins in peach, and reveals molecular basis for NH₄⁺ uptake and N nutrition mechanisms of fruit trees.     

Genome-Wide Identification,Evolution and Expression Analyses of <i>GA2ox</i> Gene Family in <i>Brassica napus</i> L.

Gibberellin 2-oxidases (GA2ox) are important enzymes that maintain the balance of bioactive GAs in plants. GA2ox genes have been identified and characterized in many plants, but these genes were not investigated in Brassica napus. Here, we identified 31 GA2ox genes in B. napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes. Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm, and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons. Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups, including two C₁₉-GA2ox and two C₂₀-GA2ox clades. Group 4 is a C₂₀-GA2ox Class discovered recently. Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes. BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome. BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development, and most of them were mainly involved in abiotic responses, regulation of phytohormones and growth and development. Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons, as well as an insight into the biological functions of GA2ox family genes in B. napus.     

DDG1 and G Protein α Subunit RGA1 Interaction Regulates Plant Height and Senescence in Rice (<i>Oryza sativa</i>)

Many studies have already shown that dwarfism and moderate delayed leaf senescence positively impact rice yield, but the underlying molecular mechanism of dwarfism and leaf senescence remains largely unknown. Here, using map-based cloning, we identified an allele of DEP2, DDG1, which controls plant height and leaf senescence in rice. The ddg1 mutant displayed dwarfism, short panicles, and delayed leaf senescence. Compared with the wild-type, ddg1 was insensitive to exogenous gibberellins (GA) and brassinolide (BR). DDG1 is expressed in various organs, especially in stems and panicles. Yeast two-hybrid assay, bimolecular fluorescent complementation and luciferase complementation image assay showed that DDG1 interacts with the α-subunit of the heterotrimeric G protein. Disruption of RGA1 resulted in dwarfism, short panicles, and darker-green leaves. Furthermore, we found that ddg1 and the RGA1 mutant was more sensitive to salt treatment, suggesting that DDG1 and RGA1 are involved in regulating salt stress response in rice. Our results show that DDG1/DEP2 regulates plant height and leaf senescence through interacting with RGA1.