High-density bin-based genetic map reveals a 530-kb chromosome segment derived from wild peanut contributing to late leaf spot resistance.

KEY MESSAGE: Twenty-eight QTLs for LLS disease resistance were identified using an amphidiploid constructed mapping population, a favorable 530-kb chromosome segment derived from wild species contributes to the LLS resistance. Late leaf spot (LLS) is one of the major foliar diseases of peanut, causing serious yield loss and affecting the quality of kernel and forage. Some wild Arachis species possess higher resistance to LLS as compared with cultivated peanut; however, ploidy level differences restrict utilization of wild species. In this study, a synthetic amphidiploid (Ipadur) of wild peanuts with high LLS resistance was used to cross with Tifrunner to construct TI population. In total, 200 recombinant inbred lines were collected for whole-genome resequencing. A high-density bin-based genetic linkage map was constructed, which includes 4,809 bin markers with an average inter-bin distance of 0.43 cM. The recombination across cultivated and wild species was unevenly distributed, providing a novel recombination landscape for cultivated-wild Arachis species. Using phenotyping data collected across three environments, 28 QTLs for LLS disease resistance were identified, explaining 4.35-20.42% of phenotypic variation. The major QTL located on chromosome 14, qLLS14.1, could be consistently detected in 2021 Jiyang and 2022 Henan with 20.42% and 12.12% PVE, respectively. A favorable 530-kb chromosome segment derived from Ipadur was identified in the region of qLLS14.1, in which 23 disease resistance proteins were located and six of them showed significant sequence variations between Tifrunner and Ipadur. Allelic variation analysis indicating the 530-kb segment of wild species might contribute to the disease resistance of LLS. These associate genomic regions and candidate resistance genes are of great significance for peanut breeding programs for bringing durable resistance through pyramiding such multiple LLS resistance loci into peanut cultivars.

Genome-wide identification and expression analysis of GA20ox and GA3ox genes during pod development in peanut.

Background: Gibberellins (GAs) play important roles in regulating peanut growth and development. GA20ox and GA3ox are key enzymes involved in GA biosynthesis. These enzymes encoded by a multigene family belong to the 2OG-Fe (II) oxygenase superfamily. To date, no genome-wide comparative analysis of peanut AhGA20ox and AhGA3ox-encoding genes has been performed, and the roles of these genes in peanut pod development are not clear. Methods: A whole-genome analysis of AhGA20ox and AhGA3ox gene families in peanut was carried out using bioinformatic tools. The expression of these genes at different stage of pod development was analyzed using qRT-PCR. Results: In this study, a total of 15 AhGA20ox and five AhGA3ox genes were identified in peanut genome, which were distributed on 14 chromosomes. Phylogenetic analysis divided the GA20oxs and GA3oxs into three groups, but AhGA20oxs and AhGA3oxs in two groups. The conserved pattern of gene structure, cis-elements, and protein motifs further confirmed their evolutionary relationship in peanut. AhGA20ox and AhGA3ox genes were differential expressed at different stages of pod development. The strong expression of AhGA20ox1/AhGA20ox4, AhGA20ox12/AhGA20ox15, AhGA3ox1 and AhGA3ox4/AhGA3ox5 in S1-stage indicated that these genes could have a key role in controlling peg elongation. Furthermore, AhGA20ox and AhGA3ox also showed diverse expression patterns in different peanut tissues including leaves, main stems, flowers and inflorescences. Noticeably, AhGA20ox9/AhGA20ox11 and AhGA3o4/AhGA3ox5 were highly expressed in the main stem, whereas the AhGA3ox1 and AhGA20ox10 were strongly expressed in the inflorescence. The expression levels of AhGA20ox2/AhGA20ox3, AhGA20ox5/AhGA20ox6, AhGA20ox7/AhGA20ox8, AhGA20ox13/AhGA20ox14 and AhGA3ox2/AhGA3ox3 were high in the flowers, suggesting their involvement in flower development. These results provide a basis for deciphering the roles of AhGA20ox and AhGA3ox in peanut growth and development, especially in pod development.

Phylogenomic Analysis of Cytochrome P450 Gene Superfamily and Their Association with Flavonoids Biosynthesis in Peanut (Arachis hypogaea L.).

Cytochrome P450s (CYPs) constitute extensive enzyme superfamilies in the plants, playing pivotal roles in a multitude of biosynthetic and detoxification pathways essential for growth and development, such as the flavonoid biosynthesis pathway. However, CYPs have not yet been systematically studied in the cultivated peanuts (Arachis hypogaea L.), a globally significant cash crop. This study addresses this knowledge deficit through a comprehensive genome-wide analysis, leading to the identification of 589 AhCYP genes in peanuts. Through phylogenetic analysis, all AhCYPs were systematically classified into 9 clans, 43 gene families. The variability in the number of gene family members suggests specialization in biological functions. Intriguingly, both tandem duplication and fragment duplication events have emerged as pivotal drivers in the evolutionary expansion of the AhCYP superfamily. Ka/Ks analysis underscored the substantial influence of strong purifying selection on the evolution of AhCYPs. Furthermore, we selected 21 genes encoding 8 enzymes associated with the flavonoid pathway. The results of quantitative real-time PCR (qRT-PCR) experiments unveiled stage-specific expression patterns during the development of peanut testa, with discernible variations between pink and red testa. Importantly, we identified a direct correlation between gene expression levels and the accumulation of metabolites. These findings offer valuable insights into elucidating the comprehensive functions of AhCYPs and the underlying mechanisms governing the divergent accumulation of flavonoids in testa of different colors.

Transcriptional networks orchestrating red and pink testa color in peanut.

BACKGROUND: Testa color is an important trait of peanut (Arachis hypogaea L.) which is closely related with the nutritional and commercial value. Pink and red are main color of peanut testa. However, the genetic mechanism of testa color regulation in peanut is not fully understood. To elucidate a clear picture of peanut testa regulatory model, samples of pink cultivar (Y9102), red cultivar (ZH12), and two RNA pools (bulk red and bulk pink) constructed from F4 lines of Y9102 x ZH12 were compared through a bulk RNA-seq approach. RESULTS: A total of 2992 differential expressed genes (DEGs) were identified among which 317 and 1334 were up-regulated and 225 and 1116 were down-regulated in the bulk red-vs-bulk pink RNA pools and Y9102-vs-ZH12, respectively. KEGG analysis indicates that these genes were divided into significantly enriched metabolic pathways including phenylpropanoid, flavonoid/anthocyanin, isoflavonoid and lignin biosynthetic pathways. Notably, the expression of the anthocyanin upstream regulatory genes PAL, CHS, and CHI was upregulated in pink and red testa peanuts, indicating that their regulation may occur before to the advent of testa pigmentation. However, the differential expression of down-stream regulatory genes including F3H, DFR, and ANS revealed that deepening of testa color not only depends on their gene expression bias, but also linked with FLS inhibition. In addition, the down-regulation of HCT, IFS, HID, 7-IOMT, and I2'H genes provided an alternative mechanism for promoting anthocyanin accumulation via perturbation of lignin and isoflavone pathways. Furthermore, the co-expression module of MYB, bHLH, and WRKY transcription factors also suggested a fascinating transcriptional activation complex, where MYB-bHLH could utilize WRKY as a co-option during the testa color regulation by augmenting anthocyanin biosynthesis in peanut. CONCLUSIONS: These findings reveal candidate functional genes and potential strategies for the manipulation of anthocyanin biosynthesis to improve peanut varieties with desirable testa color.

Insights into the Novel FAD2 Gene Regulating Oleic Acid Accumulation in Peanut Seeds with Different Maturity.

AhFAD2 is a key enzyme catalyzing the conversion of oleic acid into linoleic acid. The high oleic acid characteristic of peanut mainly comes from the homozygous recessive mutation of AhFAD2A and AhFAD2B genes (aabb). However, even in high-oleic-acid varieties with the aabb genotype, the oleic acid content of seeds with different maturity varies significantly. Therefore, in addition to AhFAD2A and AhFAD2B, other FAD2 members or regulators may be involved in this process. Which FAD2 genes are involved in the regulatory processes associated with seed maturity is still unclear. In this study, four stable lines with different genotypes (AABB, aaBB, AAbb, and aabb) were used to analyze the contents of oleic acid and linoleic acid at different stages of seed development in peanut. Three new AhFAD2 genes (AhFAD2-7, AhFAD2-8, and AhFAD2-9) were cloned based on the whole-genome sequencing results of cultivated peanuts. All peanut FAD2 genes showed tissue preference in expression; however, only the expression level of AhFAD2-7 was positively correlated with the linoleic acid concentration in peanut seeds. These findings provide new insights into the regulation of oleic acid accumulation by maturity, and AhFAD2-7 plays an important role in the maturity dependent accumulation of oleic acid and linoleic acid in peanut.

De novo full length transcriptome analysis and gene expression profiling to identify genes involved in phenylethanol glycosides biosynthesis in Cistanche tubulosa.

BACKGROUND: The dried stem of Cistanche, is a famous Chinese traditional medicine. The main active pharmacodynamic components are phenylethanol glycosides (PhGs). Cistanche tubulosa produces higher level of PhGs in its stems than that of Cistanche deserticola. However, the key genes in the PhGs biosynthesis pathway is not clear in C. tubulosa. RESULTS: In this study, we performed the full-length transcriptome sequencing and gene expression profiling of C. tubulosa using PacBio combined with BGISEQ-500 RNA-seq technology. Totally, 237,772 unique transcripts were obtained, ranging from 199 bp to 31,857 bp. Among the unique transcripts, 188,135 (79.12%) transcripts were annotated. Interestingly, 1080 transcripts were annotated as 22 enzymes related to PhGs biosynthesis. We measured the content of echinacoside, acteoside and total PhGs at two development stages, and found that the content of PhGs was 46.74% of dry matter in young fleshy stem (YS1) and then decreased to 31.22% at the harvest stage (HS2). To compare with YS1, 13,631 genes were up-regulated, and 15,521 genes were down regulated in HS2. Many differentially expressed genes (DEGs) were identified to be involved in phenylpropanoid biosynthesis pathway, phenylalanine metabolism pathway, and tyrosine metabolism pathway. CONCLUSIONS: This is the first report of transcriptome study of C. tubulosa which provided the foundation for understanding of PhGs biosynthesis. Based on these results, we proposed a potential model for PhGs biosynthesis in C. tubulosa.

Targeted metabolome analysis reveals accumulation of metabolites in testa of four peanut germplasms.

Cultivated peanut (Arachis hypogaea L.) is an important source of edible oil and protein. Peanut testa (seed coat) provides protection for seeds and serves as a carrier for diversity metabolites necessary for human health. There is significant diversity available for testa color in peanut germplasms. However, the kinds and type of metabolites in peanut testa has not been comprehensively investigated. In this study, we performed metabolite profiling using UPLC-MS/MS for four peanut germplasm lines with different testa colors, including pink, purple, red, and white. A total of 85 metabolites were identified in four peanuts. Comparative metabolomics analysis identified 78 differentially accumulated metabolites (DAMs). Some metabolites showed significant correlation with other metabolites. For instance, proanthocyanidins were positively correlated with cyanidin 3-O-rutinoside and malvin, and negatively correlated with pelargonidin-3-glucoside. We observed that the total proanthocyanidins are most abundant in pink peanut variety WH10. The red testa accumulated more isoflavones, flavonols and anthocyanidins compared with that in pink testa. These results provided valuable information about differential accumulation of metabolites in testa with different color, which are helpful for further investigation of the molecular mechanism underlying biosynthesis and accumulation of these metabolites in peanut.

BSA­seq and genetic mapping identified candidate genes for branching habit in peanut.

KEY MESSAGE: The candidate gene AhLBA1 controlling lateral branch angel of peanut was fine-mapped to a 136.65-kb physical region on chromosome 15 using the BSA-seq and QTL mapping. Lateral branch angel (LBA) is an important plant architecture trait of peanut, which plays key role in lodging, peg soil penetration and pod yield. However, there are few reports of fine mapping and quantitative trait loci (QTLs)/cloned genes for LBA in peanut. In this project, a mapping population was constructed using a spreading variety Tifrunner and the erect variety Fuhuasheng. Through bulked segregant analysis sequencing (BSA-seq), a major gene related to LBA, named as AhLBA1, was preliminarily mapped at the region of Chr.15: 150-160 Mb. Then, using traditional QTL approach, AhLBA1 was narrowed to a 1.12 cM region, corresponding to a 136.65-kb physical interval of the reference genome. Of the nine genes housed in this region, three of them were involved in hormone metabolism and regulation, including one "F-box protein" and two "2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase (2OG oxygenase)" encoding genes. In addition, we found that the level of some classes of cytokinin (CK), auxin and ethylene showed significant differences between spreading and erect peanuts at the junction of main stem and lateral branch. These findings will aid further elucidation of the genetic mechanism of LBA in peanut and facilitating marker-assisted selection (MAS) in the future breeding program.

Transcriptome Analysis and Gene Expression Profiling of the Peanut Small Seed Mutant Identified Genes Involved in Seed Size Control.

Seed size is a key factor affecting crop yield and a major agronomic trait concerned in peanut (Arachis hypogaea L.) breeding. However, little is known about the regulation mechanism of peanut seed size. In the present study, a peanut small seed mutant1 (ssm1) was identified through irradiating peanut cultivar Luhua11 (LH11) using 60Coγ ray. Since the globular embryo stage, the embryo size of ssm1 was significantly smaller than that of LH11. The dry seed weight of ssm1 was only 39.69% of the wild type LH14. The seeds were wrinkled with darker seed coat. The oil content of ssm1 seeds were also decreased significantly. Seeds of ssm1 and LH11 were sampled 10, 20, and 40 days after pegging (DAP) and were used for RNA-seq. The results revealed that genes involved in plant hormones and several transcription factors related to seed development were differentially expressed at all three stages, especially at DAP10 and DAP20. Genes of fatty acid biosynthesis and late embryogenesis abundant protein were significantly decreased to compare with LH11. Interestingly, the gene profiling data suggested that PKp2 and/or LEC1 could be the key candidate genes leading to the small seed phenotype of the mutant. Our results provide valuable clues for further understanding the mechanisms underlying seed size control in peanut.

The Mechanisms Underlying Salt Resistance Mediated by Exogenous Application of 24-Epibrassinolide in Peanut.

Peanut is one of the most important oil crops in the world, the growth and productivity of which are severely affected by salt stress. 24-epibrassinolide (EBL) plays an important role in stress resistances. However, the roles of exogenous EBL on the salt tolerance of peanut remain unclear. In this study, peanut seedlings treated with 150 mM NaCl and with or without EBL spray were performed to investigate the roles of EBL on salt resistance. Under 150 mM NaCl conditions, foliar application of 0.1 µM EBL increased the activity of catalase and thereby could eliminate reactive oxygen species (ROS). Similarly, EBL application promoted the accumulation of proline and soluble sugar, thus maintaining osmotic balance. Furthermore, foliar EBL spray enhanced the total chlorophyll content and high photosynthesis capacity. Transcriptome analysis showed that under NaCl stress, EBL treatment up-regulated expression levels of genes encoding peroxisomal nicotinamide adenine dinucleotide carrier (PMP34), probable sucrose-phosphate synthase 2 (SPS2) beta-fructofuranosidase (BFRUCT1) and Na+/H+ antiporters (NHX7 and NHX8), while down-regulated proline dehydrogenase 2 (PRODH). These findings provide valuable resources for salt resistance study in peanut and lay the foundation for using BR to enhance salt tolerance during peanut production.

Bulk RNA-Seq Analysis Reveals Differentially Expressed Genes Associated with Lateral Branch Angle in Peanut.

Lateral branch angle (LBA), or branch habit, is one of the most important agronomic traits in peanut. To date, the underlying molecular mechanisms of LBA have not been elucidated in peanut. To acquire the differentially expressed genes (DEGs) related to LBA, a TI population was constructed through the hybridization of a bunch-type peanut variety Tifrunner and prostrate-type Ipadur. We report the identification of DEGs related to LBA by sequencing two RNA pools, which were composed of 45 F3 lines showing an extreme opposite bunch and prostrate phenotype. We propose to name this approach Bulk RNA-sequencing (BR-seq) as applied to several plant species. Through BR-seq analysis, a total of 3083 differentially expressed genes (DEGs) were identified, including 13 gravitropism-related DEGs, 22 plant hormone-related DEGs, and 55 transcription factors-encoding DEGs. Furthermore, we also identified commonly expressed alternatively spliced (AS) transcripts, of which skipped exon (SE) and retained intron (RI) were most abundant in the prostrate and bunch-type peanut. AS isoforms between prostrate and bunch peanut highlighted important clues to further understand the post-transcriptional regulatory mechanisms of branch angle regulation. Our findings provide not only important insights into the landscape of the regulatory pathway involved in branch angle formation but also present practical information for peanut molecular breeding in the future.

BSA­seq and genetic mapping reveals AhRt2 as a candidate gene responsible for red testa of peanut.

KEY MESSAGE: The candidate recessive gene AhRt2 responsible for red testa of peanut was identified through combined BSA-seq and linkage mapping approaches. The testa color of peanuts (Arachis hypogaea L.) is an important trait, and those with red testa are particularly popular owing to the high-anthocyanin content. However, the identification of genes underlying the regulation of the red testa trait in peanut are rarely reported. In order to fine map red testa gene, two F2:4 populations were constructed through the cross of YZ9102 (pink testa) with ZH12 (red testa) and ZH2 (red testa). Genetic analysis indicated that red testa was controlled by a single recessive gene named as AhRt2 (Red testa gene 2). Using BSA-seq approach, AhRt2 was preliminary identified on chromosome 12, which was further mapped to a 530-kb interval using 220 recombinant lines through linkage mapping. Furthermore, functional annotation, expression profiling, and the analyses of sequence variation confirmed that the anthocyanin reductase namely (Arahy.IK60LM) was the most likely candidate gene for AhRt2. It was found that a SNP in the third exon of AhRt2 altered the encoding amino acids, and was associated with red testa in peanut. In addition, a closely linked molecular marker linked with red testa trait in peanut was also developed for future studies. Our results provide valuable insight into the molecular mechanism underlying peanut testa color and present significant diagnostic marker resources for marker-assisted selected breeding in peanut.

Phosphoproteomic Profiling Reveals Early Salt-Responsive Mechanisms in Two Foxtail Millet Cultivars.

Excess soluble salts in saline soils are harmful to most plants. Understanding the biochemical responses to salts in plants and studying the salt tolerance-associated genetic resources in nature will contribute to the improvement of salt tolerance in crops. As an emerging model crop, foxtail millet (Setaria italica L.) has been regarded as a novel species for stress resistance investigation. Here, the dynamic proteomic and phosphoproteomic profiling of two foxtail millet varieties of An04 and Yugu2 with contrasting salt tolerance characteristics were investigated under salt stress. In total, 10,366 sites representing to 2,862 proteins were detected and quantified. There were 759 and 990 sites corresponding to 484 and 633 proteins identified under salinity in An04 and Yugu2, respectively, and 1,264 and 1,131 phosphorylation sites corresponding to 789 and 731 proteins were identified between these two varieties before and after salt stress, respectively. The differentially-regulated phosphoproteins (DRPPs) were mainly involved in signal transduction, regulation of gene expression, translation, ion transport, and metabolism processes. Yugu2 possessed signal perception and transduction capabilities more rapidly and had a more intense response compared with An04 upon salinity. The sucrose metabolism pathway, in particularly, might play a vital role in salt response in foxtail millet, which not only provides UDP-glucose for the cellulose synthesis and energy production, but also promotes flavonoid related synthesis to enhance the salt tolerance ability. Over-expressing the phospho-mimic sucrose synthase (SuS) (SuS S10D ) in soybean roots enhanced salt tolerance compared with over-expressing SuS lines. The knowledge of this research will shed light on elucidating the mechanisms of salt response, and pave the way for crop varieties innovation and cultivation under salinity and stresses.

Genome-wide identification of PTI1 family in Setaria italica and salinity-responsive functional analysis of SiPTI1-5.

BACKGROUND: PTI1 (Pto-interacting 1) protein kinase belongs to the receptor-like cytoplasmic kinase (RLCK) group of receptor-like protein kinases (RLK), but lack extracellular and transmembrane domains. PTI1 was first identified in tomato (Solanum lycopersicum) and named SlPTI1, which has been reported to interact with bacterial effector Pto, a serine/threonine protein kinase involved in plant resistance to bacterial disease. Briefly, the host PTI1 specifically recognizes and interacts with the bacterial effector AvrPto, which triggers hypersensitive cell death to inhibit the pathogen growth in the local infection site. Previous studies have demonstrated that PTI1 is associated with oxidative stress and hypersensitivity. RESULTS: We identified 12 putative PTI1 genes from the genome of foxtail millet (Setaria italica) in this study. Gene replication analysis indicated that both segmental replication events played an important role in the expansion of PTI1 gene family in foxtail millet. The PTI1 family members of model plants, i.e. S. italica, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), maize (Zea mays), S. lycopersicum, and soybean (Glycine max), were classified into six major categories according to the phylogenetic analysis, among which the PTI1 family members in foxtail millet showed higher degree of homology with those of rice and maize. The analysis of a complete set of SiPTI1 genes/proteins including classification, chromosomal location, orthologous relationships and duplication. The tissue expression characteristics revealed that SiPTI1 genes are mainly expressed in stems and leaves. Experimental qRT-PCR results demonstrated that 12 SiPTI1 genes were induced by multiple stresses. Subcellular localization visualized that all of foxtail millet SiPTI1s were localized to the plasma membrane. Additionally, heterologous expression of SiPTI1-5 in yeast and E. coli enhanced their tolerance to salt stress. CONCLUSIONS: Our results contribute to a more comprehensive understanding of the roles of PTI1 protein kinases and will be useful in prioritizing particular PTI1 for future functional validation studies in foxtail millet.

Integrative analyses of transcriptomics and metabolomics upon seed germination of foxtail millet in response to salinity.

Salinity stress has become an expanding threat to food security worldwide. Revealing the mechanisms of salinity tolerance in plants has immense significance. Foxtail millet (Setaria italica L.) has been regarded as a model crop for exploring mechanisms under stress, considering its extreme adaptation abilities to adverse ecologies. In present study, two foxtail millet cultivars of Yugu2 and An04 with contrasting salt tolerance properties were investigated through integrative analyses of transcriptomics and metabolomics. In the transcriptomics results, 8887 and 12,249 DEGs were identified in Yugu2 and An04 in response to salinity, respectively, and 3149 of which were overlapped between two varieties. These salinity-responsive genes indicated that ion transport, redox homeostasis, phytohormone metabolism, signaling and secondary metabolism were enriched in Yugu2 by GO and KEGG analyses. The integrative omics analysis implied that phenylpropanoid, flavonoid and lignin biosynthesis pathways, and lysophospholipids were vital in determining the foxtail millet salinity tolerance. Importantly, the tolerance of Yugu2 attributed to higher efficiencies of ion channel and antioxidant system. All these provide a comprehensive regulatory network of foxtail millet to cope with salinity, and shed some lights on salt tolerance which is relevant for other cereal crops.

An S-domain receptor-like kinase, OsESG1, regulates early crown root development and drought resistance in rice.

Plant receptor-like kinase (RLKs) are serine/threonine protein kinases that play fundamental roles in development, innate immunity, and abiotic stress response. Here, we identified an S-domain receptor-like kinase OsESG1 from rice (Oryza sativa), and identified its involvement in early crown root (CR) development and drought response. The OsESG1 kinase domain possessed auto-phosphorylation activity and was able to phosphorylate MBP and His proteins. OsESG1 was expressed ubiquitously in all tissues that were examined, with relatively higher expression in the embryo. And it could be induced to express by treating with PEG, NaCl and ABA. Transgenic plants carrying anti-sense (AS) OsESG1 were generated by knockdown of OsESG1 expression. At the early seedling stage, AS lines had fewer CRs and shorter shoot compared with wild type (WT) plants. IAA flux and the genes' expressions of the auxin responsive and efflux carrier were infected in the AS lines. These results indicated that auxin signaling and polar auxin transport (PAT) were disrupted. The AS lines were more sensitive to osmotic stress compared to WT, and showed excessive accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), lower activities of antioxidant enzymes, and impaired expressions of stress-related genes under PEG treatment. Results above suggested that OsESG1 may regulate CR initiation and development by controlling auxin response and distribution, and participate in stress response by regulating the activities of antioxidants and expressions of stress-regulated genes.

Comparative proteomic investigation of drought responses in foxtail millet.

BACKGROUND: Foxtail millet (Setaria italica L. P. Beauv) has been considered as a tractable model crop in recent years due to its short growing cycle, lower amount of repetitive DNA, inbreeding nature, small diploid genome, and outstanding abiotic stress-tolerance characteristics. With modern agriculture facing various adversities, it's urgent to dissect the mechanisms of how foxtail millet responds and adapts to drought and stress on the proteomic-level. RESULTS: In this research, a total of 2474 differentially expressed proteins were identified by quantitative proteomic analysis after subjecting foxtail millet seedlings to drought conditions. 321 of these 2474 proteins exhibited significant expression changes, including 252 up-regulated proteins and 69 down-regulated proteins. The resulting proteins could then be divided into different categories, such as stress and defense responses, photosynthesis, carbon metabolism, ROS scavenging, protein synthesis, etc., according to Gene Ontology annotation. Proteins implicated in fatty acid and amino acid metabolism, polyamine biosynthesis, hormone metabolism, and cell wall modifications were also identified. These obtained differential proteins and their possible biological functions under drought stress all suggested that various physiological and metabolic processes might function cooperatively to configure a new dynamic homeostasis in organisms. The expression patterns of five drought-responsive proteins were further validated using western blot analysis. The qRT-PCR was also carried out to analyze the transcription levels of 21 differentially expressed proteins. The results showed large inconsistency in the variation between proteins and the corresponding mRNAs, which showed once again that post-transcriptional modification performs crucial roles in regulating gene expression. CONCLUSION: The results offered a valuable inventory of proteins that may be involved in drought response and adaption, and provided a regulatory network of different metabolic pathways under stress stimulation. This study will illuminate the stress tolerance mechanisms of foxtail millet, and shed some light on crop germplasm breeding and innovation.

Cloning and functional analysis of the SiRLK35 gene in Setaria italic L.

Receptor like protein kinases (RLKs) play vital roles in both plant development and stress conditions. Using drought-treated "Yugu 1" as materials, a drought-responsive RLK gene, SiRLK35, was isolated through iTRAQ analysis. In this study, the further analyses of the gene functions were carried out. First, full-length SiRLK35 was amplified by PCR using the cDNA of foxtail millet seedlings as a template. The expression patterns of SiRLK35 under NaCl, PEG, ABA, GA and MeJA treatment were analyzed by quantitative real-time PCR (qRT-PCR), and we found that the expression of SiRLK35 could be induced under different treatments, especially under NaCl treatment. Second, the prokaryotic expression plasmid of SiRLK35 was constructed, and the salt resistance of SiRLK35 was detected by the bacterial plaque growth method. And we uncovered that the growth and tolerance of SiRLK35-containing Escherichia coli strains were in better conditions than control under the NaCl stress. Lastly, pCambia1301P-SiRLK35 was constructed and transformed into rice to obtain transgenic plants. The tolerance of transgenic rice plants to salt stress was higher than that of controls through physiological analysis. We propose that SiRLK35 may participate in salt and stress resistance processes, which could provide potential theoretical foundation for the stress resistance varieties cultivation and breeding of foxtail millet.

Evaluating the Effect of Expressing a Peanut Resveratrol Synthase Gene in Rice.

Resveratrol (Res) is a type of natural plant stilbenes and phytoalexins that only exists in a few plant species. Studies have shown that the Res could be biosynthesized and accumulated within plants, once the complete metabolic pathway and related enzymes, such as the key enzyme resveratrol synthase (RS), existed. In this study, a RS gene named PNRS1 was cloned from the peanut, and the activity was confirmed in E. coli. Using transgenic approach, the PNRS1 transgenic rice was obtained. In T3 generation, the Res production and accumulation were further detected by HPLC. Our data revealed that compared to the wild type rice which trans-resveratrol was undetectable, in transgenic rice, the trans-resveratrol could be synthesized and achieved up to 0.697 µg/g FW in seedlings and 3.053 µg/g DW in seeds. Furthermore, the concentration of trans-resveratrol in transgenic rice seedlings could be induced up to eight or four-fold higher by ultraviolet (UV-C) or dark, respectively. Simultaneously, the endogenous increased of Res also showed the advantages in protecting the host plant from UV-C caused damage or dark-induced senescence. Our data indicated that Res was involved in host-defense responses against environmental stresses in transgenic rice. Here the results describes the processes of a peanut resveratrol synthase gene transformed into rice, and the detection of trans-resveratrol in transgenic rice, and the role of trans-resveratrol as a phytoalexin in transgenic rice when treated by UV-C and dark. These findings present new outcomes of transgenic approaches for functional genes and their corresponding physiological functions, and shed some light on broadening available resources of Res, nutritional improvement of crops, and new variety cultivation by genetic engineering.

Group 5 LEA protein, ZmLEA5C, enhance tolerance to osmotic and low temperature stresses in transgenic tobacco and yeast.

Group 5 LEA (Late Embryogenesis Abundant) proteins contain a significantly higher proportion of hydrophobic residues but lack significant signature motifs or consensus sequences. This group is considered as an atypical group of LEA proteins. Up to now, there is little known about group 5C LEA proteins in maize. Here, we identified a novel group 5C LEA protein from maize. The accumulation of transcripts demonstrated that ZmLEA5C displayed similar induced characteristics in leaves and roots. Transcription of ZmLEA5C could be induced by low temperature, osmotic and oxidative stress and some signaling molecules, such as abscisic acid (ABA), salicylic acid (SA) and methyl jasmonate (MeJA). However, transcription of ZmLEA5C was significantly inhibited by high salinity. Further study indicated that the ZmLEA5C protein could be phosphorylated by the protein kinase CKII. ZmLEA5C could protect the activity of LDH under water deficit and low temperature stresses. Overexpression of ZmLEA5C conferred to transgenic tobacco (Nicotiana benthamiana) and yeast (GS115) tolerance to osmotic and low temperature stresses.