Global analysis of seed transcriptomes reveals a novel PLATZ regulator for seed size and weight control in soybean.

Seed size and weight are important factors that influence soybean yield. Combining the weighted gene co-expression network analysis (WGCNA) of 45 soybean accessions and gene dynamic changes in seeds at seven developmental stages, we identified candidate genes that may control the seed size/weight. Among these, a PLATZ-type regulator overlapping with 10 seed weight QTLs was further investigated. This zinc-finger transcriptional regulator, named as GmPLATZ, is required for the promotion of seed size and weight in soybean. The GmPLATZ may exert its functions through direct binding to the promoters and activation of the expression of cyclin genes and GmGA20OX for cell proliferation. Overexpression of the GmGA20OX enhanced seed size/weight in soybean. We further found that the GmPLATZ binds to a 32-bp sequence containing a core palindromic element AATGCGCATT. Spacing of the flanking sequences beyond the core element facilitated GmPLATZ binding. An elite haplotype Hap3 was also identified to have higher promoter activity and correlated with higher gene expression and higher seed weight. Orthologues of the GmPLATZ from rice and Arabidopsis play similar roles in seeds. Our study reveals a novel module of GmPLATZ-GmGA20OX/cyclins in regulating seed size and weight and provides valuable targets for breeding of crops with desirable agronomic traits.

MsNRAMP2 Enhances Tolerance to Iron Excess Stress in Nicotiana tabacum and MsMYB Binds to Its Promoter.

Iron(Fe) is a trace metal element necessary for plant growth, but excess iron is harmful to plants. Natural resistance-associated macrophage proteins (NRAMPs) are important for divalent metal transport in plants. In this study, we isolated the MsNRAMP2 (MN_547960) gene from alfalfa, the perennial legume forage. The expression of MsNRAMP2 is specifically induced by iron excess. Overexpression of MsNRAMP2 conferred transgenic tobacco tolerance to iron excess, while it conferred yeast sensitivity to excess iron. Together with the MsNRAMP2 gene, MsMYB (MN_547959) expression is induced by excess iron. Y1H indicated that the MsMYB protein could bind to the "CTGTTG" cis element of the MsNRAMP2 promoter. The results indicated that MsNRAMP2 has a function in iron transport and its expression might be regulated by MsMYB. The excess iron tolerance ability enhancement of MsNRAMP2 may be involved in iron transport, sequestration, or redistribution.

Zinc-finger protein GmZF351 improves both salt and drought stress tolerance in soybean.

Abiotic stress is one of the most important factors reducing soybean yield. It is essential to identify regulatory factors contributing to stress responses. A previous study found that the tandem CCCH zinc-finger protein GmZF351 is an oil level regulator. In this study, we discovered that the GmZF351 gene is induced by stress and that the overexpression of GmZF351 confers stress tolerance to transgenic soybean. GmZF351 directly regulates the expression of GmCIPK9 and GmSnRK, leading to stomata closing, by binding to their promoter regions, which carry two CT(G/C)(T/A)AA elements. Stress induction of GmZF351 is mediated through reduction in the H3K27me3 level at the GmZF351 locus. Two JMJ30-demethylase-like genes, GmJMJ30-1 and GmJMJ30-2, are involved in this demethylation process. Overexpression of GmJMJ30-1/2 in transgenic hairy roots enhances GmZF351 expression mediated by histone demethylation and confers stress tolerance to soybean. Yield-related agronomic traits were evaluated in stable GmZF351-transgenic plants under mild drought stress conditions. Our study reveals a new mode of GmJMJ30-GmZF351 action in stress tolerance, in addition to that of GmZF351 in oil accumulation. Manipulation of the components in this pathway is expected to improve soybean traits and adaptation under unfavorable environments.

GmJAZ3 interacts with GmRR18a and GmMYC2a to regulate seed traits in soybean.

Seed weight is usually associated with seed size and is one of the important agronomic traits that determine yield. Understanding of seed weight control is limited, especially in soybean plants. Here we show that Glycine max JASMONATE-ZIM DOMAIN 3 (GmJAZ3), a gene identified through gene co-expression network analysis, regulates seed-related traits in soybean. Overexpression of GmJAZ3 promotes seed size/weight and other organ sizes in stable transgenic soybean plants likely by increasing cell proliferation. GmJAZ3 interacted with both G. max RESPONSE REGULATOR 18a (GmRR18a) and GmMYC2a to inhibit their transcriptional activation of cytokinin oxidase gene G. max CYTOKININ OXIDASE 3-4 (GmCKX3-4), which usually affects seed traits. Meanwhile, the GmRR18a binds to the promoter of GmMYC2a and activates GmMYC2a gene expression. In GmJAZ3-overexpressing soybean seeds, the protein contents were increased while the fatty acid contents were reduced compared to those in the control seeds, indicating that the GmJAZ3 affects seed size/weight and compositions. Natural variation in JAZ3 promoter region was further analyzed and Hap3 promoter correlates with higher promoter activity, higher gene expression and higher seed weight. The Hap3 promoter may be selected and fixed during soybean domestication. JAZ3 orthologs from other plants/crops may also control seed size and weight. Taken together, our study reveals a novel molecular module GmJAZ3-GmRR18a/GmMYC2a-GmCKXs for seed size and weight control, providing promising targets during soybean molecular breeding for better seed traits.

Genome-Wide Association Study of Partial Resistance to P. sojae in Wild Soybeans from Heilongjiang Province, China.

Phytophthora root rot (PRR) is a destructive disease of soybeans (Glycine max (L.) Merr) caused by Phytophthora sojae (P. sojae). The most effective way to prevent the disease is growing resistant or tolerant varieties. Partial resistance provides a more durable resistance against the pathogen compared to complete resistance. Wild soybean (Glycine soja Sieb. & Zucc.) seems to be an extraordinarily important gene pool for soybean improvement due to its high level of genetic variation. In this study, 242 wild soybean germplasms originating from different regions of Heilongjiang province were used to identify resistance genes to P. sojae race 1 using a genome-wide association study (GWAS). A total of nine significant SNPs were detected, repeatedly associated with P. sojae resistance and located on chromosomes 1, 10, 12, 15, 17, 19 and 20. Among them, seven favorable allelic variations associated with P. sojae resistance were evaluated by a t-test. Eight candidate genes were predicted to explore the mechanistic hypotheses of partial resistance, including Glysoja.19G051583, which encodes an LRR receptor-like serine/threonine protein kinase protein, Glysoja.19G051581, which encodes a receptor-like cytosolic serine/threonine protein kinase protein. These findings will provide additional insights into the genetic architecture of P. sojae resistance in a large sample of wild soybeans and P. sojae-resistant breeding through marker-assisted selection.

Selection for a Zinc-Finger Protein Contributes to Seed Oil Increase during Soybean Domestication.

Seed oil is a momentous agronomical trait of soybean (Glycine max) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351, encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1, BIOTIN CARBOXYL CARRIER PROTEIN2, 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III, DIACYLGLYCEROL O-ACYLTRANSFERASE1, and OLEOSIN2 in transgenic Arabidopsis (Arabidopsis thaliana) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean (Glycine soja) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops.

The transcriptomic signature of developing soybean seeds reveals the genetic basis of seed trait adaptation during domestication.

Cultivated soybean has undergone many transformations during domestication. In this paper we report a comprehensive assessment of the evolution of gene co-expression networks based on the analysis of 40 transcriptomes from developing soybean seeds in cultivated and wild soybean accessions. We identified 2680 genes that are differentially expressed during seed maturation and established two cultivar-specific gene co-expression networks. Through analysis of the two networks and integration with quantitative trait locus data we identified two potential key drivers for seed trait formation, GA20OX and NFYA. GA20OX encodes an enzyme in a rate-limiting step of gibberellin biosynthesis, and NFYA encodes a transcription factor. Overexpression of GA20OX and NFYA enhanced seed size/weight and oil content, respectively, in seeds of transgenic plants. The two genes showed significantly higher expression in cultivated than in wild soybean, and the increases in expression were associated with genetic variations in the promoter region of each gene. Moreover, the expression of GA20OX and NFYA in seeds of soybean accessions correlated with seed weight and oil content, respectively. Our study reveals transcriptional adaptation during soybean domestication and may identify a mechanism of selection by expression for seed trait formation, providing strategies for future breeding practice.

GmWRKY27 interacts with GmMYB174 to reduce expression of GmNAC29 for stress tolerance in soybean plants.

Soybean (Glycine max) is an important crop for oil and protein resources worldwide. The molecular mechanism of the abiotic stress response in soybean is largely unclear. We previously identified multiple stress-responsive WRKY genes from soybean. Here, we further characterized the roles of one of these genes, GmWRKY27, in abiotic stress tolerance using a transgenic hairy root assay. GmWRKY27 expression was increased by various abiotic stresses. Over-expression and RNAi analysis demonstrated that GmWRKY27 improves salt and drought tolerance in transgenic soybean hairy roots. Measurement of physiological parameters, including reactive oxygen species and proline contents, supported this conclusion. GmWRKY27 inhibits expression of a downstream gene GmNAC29 by binding to the W-boxes in its promoter region. The GmNAC29 is a negative factor of stress tolerance as indicated by the performance of transgenic hairy roots under stress. GmWRKY27 interacts with GmMYB174, which also suppresses GmNAC29 expression and enhances drought stress tolerance. The GmWRKY27 and GmMYB174 may have evolved to bind to neighbouring cis elements in the GmNAC29 promoter to co-reduce promoter activity and gene expression. Our study discloses a valuable mechanism in soybean for regulation of the stress response by two associated transcription factors. Manipulation of these genes should facilitate improvements in stress tolerance in soybean and other crops.

Alfalfa MsbHLH115 confers tolerance to cadmium stress through activating the iron deficiency response in Arabidopsis thaliana.

Cadmium (Cd) pollution severely affects plant growth and development, posing risks to human health throughout the food chain. Improved iron (Fe) nutrients could mitigate Cd toxicity in plants, but the regulatory network involving Cd and Fe interplay remains unresolved. Here, a transcription factor gene of alfalfa, MsbHLH115 was verified to respond to iron deficiency and Cd stress. Overexpression of MsbHLH115 enhanced tolerance to Cd stress, showing better growth and less ROS accumulation in Arabidopsis thaliana. Overexpression of MsbHLH115 significantly enhanced Fe and Zn accumulation and did not affect Cd, Mn, and Cu concentration in Arabidopsis. Further investigations revealed that MsbHLH115 up-regulated iron homeostasis regulation genes, ROS-related genes, and metal chelation and detoxification genes, contributing to attenuating Cd toxicity. Y1H, EMSA, and LUC assays confirmed the physical interaction between MsbHLH115 and E-box, which is present in the promoter regions of most of the above-mentioned iron homeostasis regulatory genes. The transient expression experiment showed that MsbHLH115 interacted with MsbHLH121pro. The results suggest that MsbHLH115 may directly regulate the iron-deficiency response system and indirectly regulate the metal detoxification response mechanism, thereby enhancing plant Cd tolerance. In summary, enhancing iron accumulation through transcription factor regulation holds promise for improving plant tolerance to Cd toxicity, and MsbHLH115 is a potential candidate for addressing Cd toxicity issues.

Large-scale analysis of phosphorylated proteins in maize leaf.

Phosphorylation is an ubiquitous regulatory mechanism governing the activity, subcellular localization, and intermolecular interactions of proteins. To identify a broad range of phosphoproteins from Zea mays, we enriched phosphopeptides from Zea mays leaves using titanium dioxide microcolumns and then extensively fractionated and identified the phosphopeptides by mass spectrometry. A total of 165 unique phosphorylation sites with a putative role in biological processes were identified in 125 phosphoproteins. Most of these proteins are involved in metabolism, including carbohydrate and protein metabolism. We identified novel phosphorylation sites on translation initiation factors, splicing factors, nucleolar RNA helicases, and chromatin-remodeling proteins such as histone deacetylases. Intriguingly, we also identified phosphorylation sites on several proteins associated with photosynthesis, and we speculate that these sites may be involved in carbohydrate metabolism or electron transport. Among these phosphoproteins, phosphoenolpyruvate carboxylase and NADH: nitrate reductase (NR) which catalyzes the rate-limiting and regulated step in the pathway of inorganic nitrogen assimilation were identified. A conserved phosphorylation site was found in the cytochrome b5 heme-binding domain of NADH: nitrate reductase, suggesting that NADH: nitrate reductase is phosphorylated by the same protein kinase or highly related kinases. These data demonstrate that the pathways that regulate diverse processes in plants are major targets of phosphorylation.

Comparative temporal analyses of the Pinus sylvestris L. var. mongolica litv. apical bud proteome from dormancy to growth.

Bud dormancy in perennial plants adapts to environmental and seasonal changes. Bud dormancy is of ecological interest because it affects forest population growth characteristics and is of economical interest because it impacts wood production levels. To understand Pinus sylvestris L. var. mongolica litv. bud-dormancy and bud-burst mechanisms, we characterized the proteomes of their apical buds at the four critical stages that occur during the dormancy-to-growth transition. Ninety-six proteins with altered expression patterns were identified using NanoLC-ESI-MS/MS. The majority of these proteins (57%) are involved in metabolic and other cellular processes. For 28% of the proteins, a function could not be assigned. However, because their expression levels changed, they may be potential candidate bud development- or dormancy-related proteins. Of the 75 non-redundant bud proteins identified, ascorbate peroxidase, pathogenesis-related protein PR-10, and heat shock proteins dramatically increased during August and November, suggesting that they may involved in the initiation of bud dormancy. Conversely, S-adenosylmethionine synthetase, abscisic acid/stress-induced proteins, superoxide dismutase (SOD), caffeoyl-CoA O-methyltransferase, actin, and type IIIa membrane protein cp-wap13 had greater expression levels during April, suggesting that they may be involved in the initiation of bud dormancy-release. Cell division cycle protein 48 and eukaryotic initiation factors 4A-15 and 4A had greater expression levels during May, suggesting that they may regulate cell proliferate and differentiation in the shoot apical meristem. These observations provide insights into the molecular mechanisms that induce or break bud dormancy.

Identification of low abundance polyA-binding proteins in Arabidopsis chloroplast using polyA-affinity column.

Proteins could be well separated and further identified by the use of 2-DE and related techniques. Yet, there are many proteins could not be detected even by more effective dyes because of their inherent low abundance or their low resolution. As a result, polyA-affinity column was used as a method to enrich polyA-binding proteins and then identified by MALDI-TOF-MS. In this study, 23 Arabidopsis chloroplast protein spots coded by 18 genes were identified, and majority of these proteins were classified into three related categories according to their annotations in the Swiss-Prot database, including NAD-, RNA-, and ATP-binding motifs, respectively. The major goal of the present Arabidopsis chloroplast proteomics project was to identify novel polyA-binding proteins or protein isoforms located in Arabidopsis chloroplasts and the specific research of cellular proteins with extremely low transcription levels could be fulfilled.

Identification and expression profiles analysis of odorant-binding proteins in soybean aphid, Aphis glycines (Hemiptera: Aphididae).

The soybean aphid, Aphis glycines, is an extreme specialist and an important invasive pest that relies on olfaction for behaviors such as feeding, mating, and foraging. Odorant-binding proteins (OBPs) play a vital role in olfaction by binding to volatile compounds and by regulating insect sensing of the environment. In this work we used rapid amplification of complementary DNA ends technology to identify and characterize 10 genes encoding A. glycines OBPs (AglyOBPs) belonging to 3 subfamilies, including 4 classic OBPs, 5 Plus-C OBPs, and one Minus-C OBP. Quantitative real-time polymerase chain reaction demonstrated variable specific expression patterns for the 10 genes based on developmental stage and aphid tissue sampled. Expression levels of 7 AglyOBPs (2, 3, 4, 5, 7, 9, and 10) were highest in the 4th instar, indicating that the 4th nymphal instar is an important developmental period during which soybean aphids regulate feeding and search for host plants. Tissue-specific expression results demonstrated that AglyOBP2, 7, and 9 exhibited significantly higher expression levels in antennae. Meanwhile, ligand-binding analysis of 5 OBPs demonstrated binding of AglyOBP2 and AglyOBP3 to a broad spectrum of volatiles released by green leaf plants, with bias toward 6- to 8-carbon chain volatiles and strong binding of AglyOBP7 to trans-ß-farnesene. Taken together, our findings build a foundation of knowledge for use in the study of molecular olfaction mechanisms and provide insights to guide future soybean aphid research.

Comparative proteomic analysis of apomictic monosomic addition line of Beta corolliflora and Beta vulgaris L. in sugar beet.

Apomixis refers to a process in which plants produce seed without fertilization through female syngamy that produces embryos genetically identical to the maternal parent. In sugar beet, interspecific hybrids between diploid Beta vulgaris and tetraploid Beta corolliflora were established and monosomic addition line M14 was selected because of the apomictic phenotype. By using two-dimensional electrophoresis gels we identified the proteins which were differently expressed between the M14 and B. vulgaris. A total of 27 protein spots which varied expressed between lines were isolated and successfully identified with MALDI-TOF MS. Among them five protein spots were found to be only presented in M14 and two protein spots only expressed in Beta. According to their functional annotations described in Swissprot database, these proteins were, respectively, involved in important biological pathways, such as cell division, functionally classified using the KEGG functional classification system. The result may be useful for us to better understand the genetic mechanism of apomixes.

A PP2C-1 Allele Underlying a Quantitative Trait Locus Enhances Soybean 100-Seed Weight.

Cultivated soybeans may lose some useful genetic loci during domestication. Introgression of genes from wild soybeans could broaden the genetic background and improve soybean agronomic traits. In this study, through whole-genome sequencing of a recombinant inbred line population derived from a cross between a wild soybean ZYD7 and a cultivated soybean HN44, and mapping of quantitative trait loci for seed weight, we discovered that a phosphatase 2C-1 (PP2C-1) allele from wild soybean ZYD7 contributes to the increase in seed weight/size. PP2C-1 may achieve this function by enhancing cell size of integument and activating a subset of seed trait-related genes. We found that PP2C-1 is associated with GmBZR1, a soybean ortholog of Arabidopsis BZR1, one of key transcription factors in brassinosteroid (BR) signaling, and facilitate accumulation of dephosphorylated GmBZR1. In contrast, the PP2C-2 allele with variations of a few amino acids at the N-terminus did not exhibit this function. Moreover, we showed that GmBZR1 could promote seed weight/size in transgenic plants. Through analysis of cultivated soybean accessions, we found that 40% of the examined accessions do not have the PP2C-1 allele, suggesting that these accessions can be improved by introduction of this allele. Taken together, our study identifies an elite allele PP2C-1, which can enhance seed weight and/or size in soybean, and pinpoints that manipulation of this allele by molecular-assisted breeding may increase production in soybean and other legumes/crops.