Natural resistance-associated macrophage proteins are involved in tolerance to heavy metal Cd2+ toxicity and resistance to bacterial wilt of peanut (Arachis hypogaea L.).

Peanut (Arachis hypogaea L.) is one of the most important oil and industrial crops. However, heavy-metal pollution and frequent soil diseases, poses a significant threat to the production of green and healthy peanuts. Herein, we investigated the effects of heavy metal Cd2+ toxicity to the peanuts, and screened out two peanut cultivars H108 and YZ 9102 with higher Cd2+-tolerance. RNA-seq revealed that Natural resistance-associated macrophage proteins (NRAMP)-like genes were involved in the Cd2+ stress tolerance in H108. Genome-wide identification revealed that 28, 13 and 9 Nramp-like genes existing in the A. hypogaea, A. duranensis and A. ipaensis, respectively. The 50 peanut NRAMP genes share conserved architectural characters, and they were classified into two groups. Expressions of AhNramps, particularly AhNramp4, AhNramp12, AhNramp19, and AhNramp25 could be greatly induced by not only cadmium toxicity, but also copper and zinc stresses. The expression profiles of AhNramp14, AhNramp16 and AhNramp25 showed significant differences in the H108 (resistance) and H107 (susceptible) under the infection of bacterial wilt. In addition, we found that the expression profiles of AhNramp14, AhNramp16, and AhNramp25 were greatly up- or down-regulated by the application of exogenous salicylic acid, methyl jasmonate, and abscisic acid. The AhNramp25, of which expression was affected by both heavy metal toxicity and bacterial wilt infection, were selected as strong candidate genes for peanut stress breeding. Our findings will provide an additional information required for further analysis of AhNramps involved in tolerance to heavy metal toxicity and resistance to bacterial wilt of peanut.

Dynamic N6 -methyladenosine RNA modification regulates peanut resistance to bacterial wilt.

N6 -methyladenosine (m6 A) is the most abundant mRNA modification in eukaryotes and is an important regulator of gene expression as well as many other critical biological processes. However, the characteristics and functions of m6 A in peanut (Arachis hypogea L.) resistance to bacterial wilt (BW) remain unknown. Here, we analyzed the dynamic of m6 A during infection of resistant (H108) and susceptible (H107) peanut accessions with Ralstonia solanacearum (R. solanacearum), the causative agent of BW. Throughout the transcriptome, we identified 'URUAY' as a highly conserved motif for m6 A in peanut. The majority of differential m6 A located within the 3' untranslated region (UTR) of the transcript, with fewer in the exons. Integrative analysis of RNA-Seq and m6 A methylomes suggests the correlation between m6 A and gene expression in peanut R. solanacearum infection, and functional analysis reveals that m6 A-associated genes were related to plant-pathogen interaction. Our experimental analysis suggests that AhALKBH15 is an m6 A demethylase in peanut, leading to decreased m6 A levels and upregulation of the resistance gene AhCQ2G6Y. The upregulation of AhCQ2G6Y expression appears to promote BW resistance in the H108 accession.

Coordinated Lipid Mobilization during Seed Development and Germination in Peanut (Arachis hypogaea L.).

Peanut (Arachis hypogaea L.) is one of the most important oil crops in the world due to its lipid-rich seeds. Lipid accumulation and degradation play crucial roles in peanut seed maturation and seedling establishment, respectively. Here, we utilized lipidomics and transcriptomics to comprehensively identify lipids and the associated functional genes that are important in the development and germination processes of a large-seed peanut variety. A total of 332 lipids were identified; triacylglycerols (TAGs) and diacylglycerols were the most abundant during seed maturation, constituting 70.43 and 16.11%, respectively, of the total lipids. Significant alterations in lipid profiles were observed throughout seed maturation and germination. Notably, TAG (18:1/18:1/18:2) and (18:1/18:2/18:2) peaked at 23386.63 and 23392.43 nmol/g, respectively, at the final stage of seed development. Levels of hydroxylated TAGs (HO-TAGs) increased significantly during the initial stage of germination. Accumulation patterns revealed an inverse relationship between free fatty acids and TAGs. Lipid degradation was determined to be regulated by diacylglycerol acyltransferase, triacylglycerol lipase, and associated transcription factors, predominantly yielding oleic acid, linoleic acid, and linolenic acid. Collectively, the results of this study provide valuable insights into lipid dynamics during the development and germination of large-seed peanuts, gene resources, and guiding future research into lipid accumulation in an economically important crop.

Dynamic DNA methylation modification in peanut seed development.

Cytosine methylation is an important epigenetic modification involved in regulation of plant development. However, the epigenetic mechanisms governing peanut seed development remain unclear. Herein, we generated DNA methylation profiles of developmental seeds of peanut H2014 and its smaller seed mutant H1314 at 15 and 60 days after pegging (DAP, S1, S4). Accompanying seed development, globally elevated methylation was observed in both lines. The mutant had a higher methylation level of 31.1% than wild type at S4, and 27.1-35.9% of the differentially methylated regions (DMRs) between the two lines were distributed in promoter or genic regions at both stages. Integrated methylome and transcriptome analysis revealed important methylation variations closely associated with seed development. Furthermore, some genes showed significantly negative correlation of expression with the methylation level within promoter or gene body. The results provide insights into the roles of DNA methylation in peanut seed development.

PSW1, an LRR receptor kinase, regulates pod size in peanut.

Pod size is a key agronomic trait that greatly determines peanut yield, the regulatory genes and molecular mechanisms that controlling peanut pod size are still unclear. Here, we used quantitative trait locus analysis to identify a peanut pod size regulator, POD SIZE/WEIGHT1 (PSW1), and characterized the associated gene and protein. PSW1 encoded leucine-rich repeat receptor-like kinase (LRR-RLK) and positively regulated pod stemness. Mechanistically, this allele harbouring a 12-bp insertion in the promoter and a point mutation in the coding region of PSW1 causing a serine-to-isoleucine (S618I) substitution substantially increased mRNA abundance and the binding affinity of PSW1 for BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). Notably, PSW1HapII (super-large pod allele of PSW1) expression led to up-regulation of a positive regulator of pod stemness PLETHORA 1 (PLT1), thereby resulting in larger pod size. Moreover, overexpression of PSW1HapII increased seed/fruit size in multiple plant species. Our work thus discovers a conserved function of PSW1 that controls pod size and provides a valuable genetic resource for breeding high-yield crops.

Multilocation proteins in organelle communication: Based on protein-protein interactions.

Protein-protein interaction (PPI) plays a crucial role in most biological processes, including signal transduction and cell apoptosis. Importantly, the knowledge of PPIs can be useful for identification of multimeric protein complexes and elucidation of uncharacterized protein functions. Arabidopsis thaliana, the best-characterized dicotyledonous plant, the steadily increasing amount of information on the levels of its proteome and signaling pathways is progressively enabling more researchers to construct models for cellular processes for the plant, which in turn encourages more experimental data to be generated. In this study, we performed an overview analysis of the 10 major organelles and their associated proteins of the dicotyledonous model plant Arabidopsis thaliana via PPI network, and found that PPI may play an important role in organelle communication. Further, multilocation proteins, especially phosphorylation-related multilocation proteins, can function as a "needle and thread" via PPIs and play an important role in organelle communication. Similar results were obtained in a monocotyledonous model crop, rice. Furthermore, we provide a research strategy for multilocation proteins by LOPIT technique, proteomics, and bioinformatics analysis and also describe their potential role in the field of plant science. The results provide a new view that the phosphorylation-related multilocation proteins play an important role in organelle communication and provide new insight into PPIs and novel directions for proteomic research. The research of phosphorylation-related multilocation proteins may promote the development of organelle communication and provide an important theoretical basis for plant responses to external stress.

Genome-wide identification and expression analysis of auxin response factors in peanut (Arachis hypogaea L.).

Auxin response factors (ARFs) are transcription factors that regulate the expression of auxin response genes, and have important functions in plant growth and development. In this study, available genome data for peanut (Arachis hypogaea L.) were used to identify AhARF genes. In total, 61 AhARFs and 23 AtARFs were divided into six groups (I-VI). Molecular structural analysis revealed that the protein members of AhARF contain at least two domains, the B3 domain and the Auxin-resp domain, and that some have a C-terminal dimerisation domain. Screening of the transcriptome data of 22 tissues of A. hypogaea cv. Tifrunner in a public database showed high expression levels of AhARF2 and AhARF6. AhARF6 was expressed more highly in the stem and branch than in the root and leaf of the wild species Arachis monticola (A. mon) and cultivated species H103. After treatment with exogenous auxin (NAA), the expression of AhARF6 was inhibited, and this inhibition was greater in A. mon than in H103. The transcriptome map revealed that the expression of AhARF6 was higher in the larger pods of H8107 and ZP06 than in the medium pods of H103 and small pods of A. mon. Moreover, AhARF6-5 was proven to be localised in the nucleus, consistent with the location of AtARF6. These results suggest that AhARF6 may play an important role in pod development in peanut.

Chromatin spatial organization of wild type and mutant peanuts reveals high-resolution genomic architecture and interaction alterations.

BACKGROUND: Three-dimensional (3D) chromatin organization provides a critical foundation to investigate gene expression regulation and cellular homeostasis. RESULTS: Here, we present the first 3D genome architecture maps in wild type and mutant allotetraploid peanut lines, which illustrate A/B compartments, topologically associated domains (TADs), and widespread chromatin interactions. Most peanut chromosomal arms (52.3%) have active regions (A compartments) with relatively high gene density and high transcriptional levels. About 2.0% of chromosomal regions switch from inactive to active (B-to-A) in the mutant line, harboring 58 differentially expressed genes enriched in flavonoid biosynthesis and circadian rhythm functions. The mutant peanut line shows a higher number of genome-wide cis-interactions than its wild-type. The present study reveals a new TAD in the mutant line that generates different chromatin loops and harbors a specific upstream AP2EREBP-binding motif which might upregulate the expression of the GA2ox gene and decrease active gibberellin (GA) content, presumably making the mutant plant dwarf. CONCLUSIONS: Our findings will shed new light on the relationship between 3D chromatin architecture and transcriptional regulation in plants.

Comprehensive Transcriptome Analyses Reveal Candidate Genes for Variation in Seed Size/Weight During Peanut (Arachis hypogaea L.) Domestication.

Seed size/weight, a key domestication trait, is also an important selection target during peanut breeding. However, the mechanisms that regulate peanut seed development are unknown. We re-sequenced 12 RNA samples from developing seeds of two cultivated peanut accessions (Lines 8106 and 8107) and wild Arachis monticola at 15, 30, 45, and 60 days past flowering (DPF). Transcriptome analyses showed that ∼36,000 gene loci were expressed in each of the 12 RNA samples, with nearly half exhibiting moderate (2 ≤ FPKM < 10) expression levels. Of these genes, 12.2% (4,523) were specifically expressed during seed development, mainly at 15 DPF. Also, ∼12,000 genes showed significant differential expression at 30, 45, and/or 60 DPF within each of the three peanut accessions, accounting for 31.8-34.1% of the total expressed genes. Using a method that combined comprehensive transcriptome analysis and previously mapped QTLs, we identified several candidate genes that encode transcription factor TGA7, topless-related protein 2, IAA-amino acid hydrolase ILR1-like 5, and putative pentatricopeptide repeat-containing (PPR) protein. Based on sequence variations identified in these genes, SNP markers were developed and used to genotype both 30 peanut landraces and a genetic segregated population, implying that EVM0025654 encoding a PPR protein may be associated with the increased seed size/weight of the cultivated accessions in comparison with the allotetraploid wild peanut. Our results provide additional knowledge for the identification and functional research into candidate genes responsible for the seed size/weight phenotype in peanut.

QTLs for constitutive aerenchyma from Zea nicaraguensis improve tolerance of maize to root-zone oxygen deficiency.

Zea nicaraguensis is a wild relative of Zea mays subsp. mays (maize) that has high waterlogging tolerance. One of its traits is constitutive aerenchyma formation (CAF) in roots and this may be one of the reasons for the tolerance, but it has not yet been proven by comparing plants that differ only in CAF in the same genetic background. We therefore produced an introgression line AE24-50-44-91 (IL-AE91) possessing four quantitative trait loci for CAF from Z. nicaraguensis in the background of maize (inbred line Mi29). The degree of root CAF in IL-AE91 was intermediate between that of Mi29 and Z. nicaraguensis. Seedlings of IL-AE91 grown aerobically were more tolerant to transfer to oxygen-deficient conditions than were Mi29 seedlings. On day 2 of oxygen deficiency, the root extension rate and viability of root-tip cells in IL-AE91 were ~2.7 and ~1.3 times greater, respectively, than they were in Mi29. On day 4, the area of aerenchyma at 80 mm from the root tips was ~1.5 times greater in IL-AE91 and radial oxygen loss from the apical parts of roots was ~3.4 times higher than in Mi29. These results demonstrate that CAF reduces the stress from low external oxygen levels caused by soil waterlogging.

Advances in crop proteomics: PTMs of proteins under abiotic stress.

Under natural conditions, crop plants are frequently subjected to various abiotic environmental stresses such as drought and heat wave, which may become more prevalent in the coming decades. Plant acclimation and tolerance to an abiotic stress are always associated with significant changes in PTMs of specific proteins. PTMs are important for regulating protein function, subcellular localization and protein activity and stability. Studies of plant responses to abiotic stress at the PTMs level are essential to the process of plant phenotyping for crop improvement. The ability to identify and quantify PTMs on a large-scale will contribute to a detailed protein functional characterization that will improve our understanding of the processes of crop plant stress acclimation and stress tolerance acquisition. Hundreds of PTMs have been reported, but it is impossible to review all of the possible protein modifications. In this review, we briefly summarize several main types of PTMs regarding their characteristics and detection methods, review the advances in PTMs research of crop proteomics, and highlight the importance of specific PTMs in crop response to abiotic stress.

Making better maize plants for sustainable grain production in a changing climate.

Achieving grain supply security with limited arable land is a major challenge in the twenty-first century, owing to the changing climate and increasing global population. Maize plays an increasingly vital role in global grain production. As a C4 plant, maize has a high yield potential. Maize is predicted to become the number one cereal in the world by 2020. However, maize production has plateaued in many countries, and hybrid and production technologies have been fully exploited. Thus, there is an urgent need to shape maize traits and architectures for increased stress tolerance and higher yield in a changing climate. Recent achievements in genomics, proteomics, and metabolomics have provided an unprecedented opportunity to make better maize. In this paper, we discuss the current challenges and potential of maize production, particularly in China. We also highlight the need for enhancing maize tolerance to drought and heat waves, summarize the elite shoot and root traits and phenotypes, and propose an ideotype for sustainable maize production in a changing climate. This will facilitate targeted maize improvement through a conventional breeding program combined with molecular techniques.

Differences in properties and proteomes of the midribs contribute to the size of the leaf angle in two near-isogenic maize lines.

The midrib of maize leaves provides the primary support for the blade and is largely associated with leaf angle size. To elucidate the role of the midrib in leaf angle formation, the maize line Shen137 (larger leaf angle) and a near isogenic line (NIL, smaller leaf angle) were used in the present study. The results of the analysis showed that both the puncture forces and proximal collenchyma number of the midribs of the first and second leaves above the ear were higher in NIL than in Shen137. Comparative proteomic analysis was performed to reveal protein profile differences in the midribs of the 5th, 10th and 19th newly expanded leaves between Shen137 and NIL. Quantitative analysis of 24 identified midrib proteins indicated that the maximum changes in abundance of 22 proteins between Shen137 and NIL appeared at the 10th leaf stage, of which phosphoglycerate kinase, adenosine kinase, fructose-bisphosphate aldolase and adenylate kinase were implicated in glycometabolism. Thus, glycometabolism might be associated with leaf angle formation and the physical and mechanical properties of the midribs. These results provide insight into the mechanism underlying maize leaf angle formation.

Diversity and function of maize pollen coat proteins: from biochemistry to proteomics.

Maize (Zea mays L.) is globally cultivated as one of the most important grain crops. As a wind-pollinated species, maize produces a large quantity of pollen grains that heavier and larger compared to Arabidopsis. Maize is an important model plant in pollen biology of monocots. The pollen coat, the outermost layer of pollen, plays a vital role in pollen-stigma interactions and successful fertilization. Pollen coat proteins (PCPs), which confer species specificity, are required for pollen adhesion, recognition, hydration, and germination on the stigma. Thus, PCPs have attracted intensive research efforts in plant science for decades. However, only a few PCPs in maize have been characterized to date, whereas the functions of most maize PCPs remain unclear. In this review, we summarize the current knowledge of maize PCPs with regard to protein constituents, synthesis and transport, and functions by comparison with the model plant Arabidopsis thaliana and Brassica plants. An understanding of the comprehensive knowledge of maize PCPs will help to illuminate the mechanism by which PCPs are involved in pollen-stigma interactions in maize and other crop plants.

Protein sHSP26 improves chloroplast performance under heat stress by interacting with specific chloroplast proteins in maize (Zea mays).

We recently demonstrated that chloroplast small HSP26 (sHSP26) is abundant in maize leaves under heat stress and potentially involved in maize heat tolerance. However, it largely remains unclear how sHSP26 functions in maize under heat stress. Here, 2-DE-based proteomics, RNA interference (RNAi), co-immunoprecipitation (Co-IP) and yeast two-hybrid (Y2H) were used to reveal chloroplast proteins interacting with sHSP26 and how sHSP26 functions under heat stress. After the silencing of sHSP26, a total of 45 protein spots from isolated protoplasts were greatly changed in abundance, of which 33 spots are chloroplastic. Co-IP revealed that nine proteins possibly associated with sHSP26. Y2H demonstrated that six chloroplast proteins interact with sHSP26 under heat stress. In particular, four proteins, including ATP synthase subunit ß, chlorophyll a-b binding protein, oxygen-evolving enhancer protein 1 and photosystem I reaction center subunit IV, strongly interacted with sHSP26 and their abundance greatly declined after RNAi of sHSP26 under heat stress. In addition, H2O2 accumulation in the chloroplasts significantly increased the expression of sHSP26, and the suppression of sHSP26 expression significantly reduced the O2 evolution rate of photosystem II under heat stress. Overall, these findings demonstrate the relevance of sHSP26 in protecting maize chloroplasts under heat stress. BIOLOGICAL SIGNIFICANCE: Maize is one of the most important crops worldwide. Frequent heat stress reduces significantly the yield and quality of maize. Our results demonstrated that sHSP26 improved maize chloroplast performance under heat stress by interacting with specific proteins. These findings are useful for understanding the mechanism of heat stress response and heat-tolerant molecular breeding in maize.

Chloroform-assisted phenol extraction improving proteome profiling of maize embryos through selective depletion of high-abundance storage proteins.

The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1) is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted phenol extraction (CAPE) method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to phenol extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize) and dicot (soybean and pea) seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and phenol extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored.