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Legume crops establish symbiosis with nitrogen-fixing rhizobia for biological nitrogen fixation (BNF), a process that provides a prominent natural nitrogen source in agroecosystems; and efficient nodulation and nitrogen fixation processes require a large amount of phosphorus (P). Here, a role of GmPAP4, a nodule-localized purple acid phosphatase, in BNF and seed yield was functionally characterized in whole transgenic soybean (Glycine max) plants under a P-limited condition. GmPAP4 was specifically expressed in the infection zones of soybean nodules and its expression was greatly induced in low P stress. Altered expression of GmPAP4 significantly affected soybean nodulation, BNF, and yield under the P-deficient condition. Nodule number, nodule fresh weight, nodule nitrogenase, APase activities, and nodule total P content were significantly increased in GmPAP4 overexpression (OE) lines. Structural characteristics revealed by toluidine blue staining showed that overexpression of GmPAP4 resulted in a larger infection area than wild-type (WT) control. Moreover, the plant biomass and N and P content of shoot and root in GmPAP4 OE lines were also greatly improved, resulting in increased soybean yield in the P-deficient condition. Taken together, our results demonstrated that GmPAP4, a purple acid phosphatase, increased P utilization efficiency in nodules under a P-deficient condition and, subsequently, enhanced symbiotic BNF and seed yield of soybean. [ABSTRACT FROM AUTHOR]

Soybean is an important economic crop in the world, which provides plant protein, oil and a variety of active substances beneficial to human health. In order to comprehensively identify the quality characters of soybean resources abundant in China, 16 kinds of seed quality traits of 520 soybean germplasms in Huang-Huai-Hai ecoregion were evaluated in 6 environments across 2 years with reference to the national standard method. The results showed that the genetic variations of the tested seed quality traits were abundant in the soybean germplasms, and the variation coefficients of isoflavone and tocopherol contents were relative high, while the variation coefficients of protein, oil and fatty acid components were relatively low. Meanwhile, there were significant correlations among the 16 seed quality traits. Principal component analysis showed that the 16 quality traits could be attributed to 5 principal components. Based on the results of principal component analysis, 520 germplasms could be divided into 3 categories by cluster analysis with significant differences. Based on the above results and high stability coefficient analysis, 35 germplasms with multiple or single elite seed quality traits were screened out, which provided important resources for genetic improvement of seed quality traits in soybean. [ABSTRACT FROM AUTHOR]

Chlorophyll is an essential pigment for plant photosynthesis. Drought tolerance affects chlorophyll synthesis, reduces photosynthetic efficiency, and decreases soybean yield. In order to explore the genetic loci of chlorophyll content in soybean under drought stress, drought resistance index (DRI) were calculated by chlorophyll contents under drought and well-watered conditions. Moreover, based on the DRI and re-sequenced genotypes of the soybean natural population (199 accessions) at 20x sequencing depth, the genome-wide association study (GWAS) was performed. The results showed that 237 single nucleotide polymorphisms (SNPs) in 18 loci on the chromosomes 1, 2, 3, 7, 10, 16, 17 and 18 were identified to associate with DRI. Moreover, 181 SNPs (76.37% of the total) were located on chromosome 16, of which 9 SNPs were located in the gene introns, 17 in exons, 21 in upstream or downstream, and 1 in 3' UTR. Based on these, 57 candidate genes flanking the associated SNPs were found, and according to the gene annotation, SNP mutation, and RNA expression, 3 candidate genes, Glyma.16G063600 (F-box protein), Glyma. 10G007000 (AP2-EREBP), and Glyma. 17G143900 (AP2/ERF) were screened out which might be responsible for the drought tolerance in soybean. Above results provided important theoretical foundation for drought tolerance genetic improvement in soybean. [ABSTRACT FROM AUTHOR]

Sugars are always transported to the other organs via sugar transporters to play their important functions in plants. SWEET (sugars will eventually be exported transporters) is a kind of sugar transporters which plays important function in plant growth and development as well as in resistance or tolerance to diverse biotic and abiotic stresses. To analyze the expressions of SWEET genes during pod and seed developments and under different stress conditions in soybean, the SWEET genes were firstly analyzed based on the newly published cultivated and wild soybean genomes in the present study. Subsequently, the gene expressions were evaluated during pod and seed developments, as well as in resistance to soybean mosaic virus (SMV) and tolerance to low phosphorus. The results showed that 48 SWEET genes were identified in the cultivated soybean genome (Wm82a4v1), which encoded protein with length 174~354 amino acids and located on 15 chromosomes. Meanwhile, 51 SWEET genes were identified in the wild soybean genome (W05), which encoded length of 84~392 amino acids and located on 16 chromosomes. The phylogenetic tree of SWEET genes in cultivated and wild soybeans showed that they were clustered into 3 sub-groups. Moreover, 16 SWEET genes were expressed in soybean pods with the expressions of Glyma. 06G122200, Glyma. 14G159900 and Glyma. 14G160100 etc. increased during the pod development process. 12 SWEET genes were expressed in soybean seeds, and the expressions of Glyma.08G025100, Glyma.13G041300 and Glyma.14G120300 were increased during the seed development, suggesting their important roles for soybean seed development. The SWEET genes showed different expressions after the inoculation of SMV, and the expressions of Glyma.08G009900 and Glyma.13G264400 were induced in leaves of resistant variety, while had no change in the sensitive variety, implying that they might involve in SMV resistance. The expressions of Glyma.04G198400, Glyma.14G160100 and Glyma.15G211800 were induced in soybean roots after low phosphorus treatment, indicating their important functions in the tolerance to low phosphorus stress. In addition, the single nucleotide polymorphism(SNP)analysis of SWEET genes showed that 43 genes contained 220 nonsynonymous SNPs with 103 located in the conserved domains of encoding proteins. Thus, above results provided candidate genes for molecular breeding of soybean pod and seed yield and diverse stress resistances. [ABSTRACT FROM AUTHOR]

Soybean provides large amounts of protein for human, and the most popular consumption pattern is tofu and soymilk. Recently, the demand of tofu and soymilk is increased, and the breeding of special variety becomes very urgent. To illustrate the genetic basis of these traits, 269 accessions were evaluated under six environments, and 32 associated SNPs were identified on nine chromosomes across multiple environments or conferring diverse traits. Of these SNPs, 16 were demonstrated by BLUP values. Seven adjacent‐SNPs were first identified to associate with tofu and soymilk weights in an 1.02‐Mb region on chromosome 8, which were verified by BLUP‐values. Meanwhile, four neighbouring SNPs in a 0.32‐Mb region on chromosome 12 were first identified to associate with these traits across multiple environments, of which three were detected in BLUP values. Furthermore, some candidate genes, that is, α‐glucan water dikinase Glyma.08G283700 and E3 SUMO‐protein ligase Glyma.12G071300, were screened out with differential expressions between the high‐ and low‐tofu/soymilk‐output accessions. Thus, these associated SNPs and candidate genes provide new insight into the genetic basis of tofu and soymilk processing quality in soybean. [ABSTRACT FROM AUTHOR]

Seed coat cracking (SCC) is a major factor that affects seed appearance quality, pathogen infection, humility absorption and seed storage in soybean. In view of this, a recombinant inbred line (RIL) population and a natural population were genotyped by SoySNP6K and were evaluated for type‐I SCC percentage. Eight QTLs were identified on six chromosomes with phenotypic variation explanations ranging from 3.46% to 34.71% in RIL population. Twenty‐three SNPs associated with cracking percentage were identified on eight chromosomes in natural population. More importantly, the association SNP ss715593768 in natural population was the left marker of qSCC‐C2‐1 in RIL population, which were detected across all of the environments and BLUP values. Two genes, Glyma.06G197300 and Glyma.06G202100, were proposed in this QTL region. These two genes showed differential expressions at seed developmental stages between RIL parents in the seed transcriptome sequencing and quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis, and also showed differential expressions between two germplasms with different cracking percentage in natural population. Thus, the results provide novel insight into the genetic basis and molecular improvement of SCC in soybean. [ABSTRACT FROM AUTHOR]

Background: Biological nitrogen fixation (BNF) is an important nitrogen source for legume plants, and highly efficient nitrogen fixation requires sufficient phosphorus (P). However, the mechanism of maintaining nitrogen fixation of the legume nodules under low P concentration remains largely unknown. Results: A nodule-localized SPX protein, GmSPX8, was discovered by transcriptome and functional analysis of its role in N2 fixation was characterized in soybean nodules. GmSPX8 was preferentially expressed in nodules and its expression was gradually increased during nodule development. And also the expression pattern was investigated using reporter gene β-glucuronidase (GUS) driven by the promoter of GmSPX8. GmSPX8 was greatly induced and the GUS activity was increased by 12.2% under P deficiency. Overexpression of GmSPX8 in transgenic plants resulted in increased nodule number, nodule fresh weight and nitrogenase activity by 15.0%, 16.0%, 42.5%, subsequently leading to increased N and P content by 17.0% and 19.0%, while suppression of GmSPX8 showed significantly impaired nodule development and nitrogen fixation efficiency under low P stress. These data indicated that GmSPX8 conferred nodule development and nitrogen fixation under low P condition. By yeast two-hybrid screening, GmPTF1 was identified as a potential interacting protein of GmSPX8, which was further confirmed by BiFC, Y2H and pull down assay. Transcript accumulation of GmPTF1 and its downstream genes such as GmEXLB1 and EXPB2 were increased in GmSPX8 overexpressed transgenic nodules, and in the presence of GmSPX8, the transcriptional activity of GmPTF1 in yeast cells and tobacco leaves was greatly enhanced. Conclusions: In summary, these findings contribute novel insights towards the role of GmSPX8 in nodule development and nitrogen fixation partly through interacting with GmPTF1 in soybean under low P condition. [ABSTRACT FROM AUTHOR]

Small heat shock proteins (sHSPs) are ubiquitous proteins present in all organisms. The sHSPs are not only upregulated under heat shock as well as other stresses but also are expressed in unstressed cells, indicating quite diverse functions of sHSPs. However, there is little known about the role of sHSPs in nodulation and nitrogen fixation in soybean. In this study, we cloned a candidate protein of sHSP, GmHSP17.1, from proteome of nodule and analyzed its function in soybean nodulation. We found that GmHSP17.1 was a cytosolic protein and preferentially expressed during nodule development. An overexpression of GmHSP17.1 in composite transgenic plants showed increases in nodule number, fresh weight, nodule size, area of infection cells, and nitrogenase activity, and subsequently promoted the content of nitrogen and growth of soybean plants. While GmHSP17.1 RNA interference (RNAi) lines showed significantly impaired nodule development and nitrogen fixation efficiency. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS), GmRIP1 was identified as the first potential target of GmHSP17.1, and was shown to be specifically expressed in soybean nodules. The interaction between GmHSP17.1 and GmRIP1 was further confirmed by yeast-two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) in vivo and pull-down assay in vitro. Furthermore, peroxidase activity was markedly increased in GmHSP17.1 overexpressed nodules and decreased in RNAi lines. As a result, the reactive oxygen species (ROS) content greatly decreased in GmHSP17.1 overexpression lines and increased in suppression lines. Taken together, we conclude that GmHSP17.1 plays an important role in soybean nodulation through interacting with GmRIP1. Our results provide foundation for studying the mechanism of nitrogen fixation and for the genetics improvement of legume plants. [ABSTRACT FROM AUTHOR]

Summary: Legume–rhizobia symbiosis enables biological nitrogen fixation to improve crop production for sustainable agriculture. Small heat shock proteins (sHSPs) are involved in multiple environmental stresses and plant development processes. However, the role of sHSPs in nodule development in soybean remains largely unknown. In the present study, we identified a nodule‐localized sHSP, called GmHSP17.9, in soybean, which was markedly up‐regulated during nodule development. GmHSP17.9 was specifically expressed in the infected regions of the nodules. GmHSP17.9 overexpression and RNAi in transgenic composite plants and loss of function in CRISPR‐Cas9 gene‐editing mutant plants in soybean resulted in remarkable alterations in nodule number, nodule fresh weight, nitrogenase activity, contents of poly β‐hydroxybutyrate bodies (PHBs), ureide and total nitrogen content, which caused significant changes in plant growth and seed yield. GmHSP17.9 was also found to act as a chaperone for its interacting partner, GmNOD100, a sucrose synthase in soybean nodules which was also preferentially expressed in the infected zone of nodules, similar to GmHSP17.9. Functional analysis of GmNOD100 in composite transgenic plants revealed that GmNOD100 played an essential role in soybean nodulation. The hsp17.9 lines showed markedly more reduced sucrose synthase activity, lower contents of UDP‐glucose and acetyl coenzyme A (acetyl‐CoA), and decreased activity of succinic dehydrogenase (SDH) in the tricarboxylic acid (TCA) cycle in nodules due to the missing interaction with GmNOD100. Our findings reveal an important role and an unprecedented molecular mechanism of sHSPs in nodule development and nitrogen fixation in soybean. [ABSTRACT FROM AUTHOR]

Seed size and shape are important characteristics in soybean breeding, whereas the genetic basis are still unknown, which constrains the improvement of related traits. In view of this, one recombinant inbred line population was assessed for six related traits under four environments. In total, 10 QTLs controlling the related traits were identified, and five were detected across at least two environments, which located on chromosomes 2, 4, 6, 13, and 16 with phenotypic variation explained (PVE) 3.6% to 9.4%. Among them, qSS‐02 and qSS‐16 were identified to control four and five related traits with pleiotropic effects on chromosomes 2 and 16 across three and two environments, respectively. Meanwhile, qSS‐04 was detected to control all of the six related traits on chromosome 4 under four environments with PVEs 4.5% to 9.4%. Furthermore, the candidate genes, that is, Glyma.04G055100 (big grain protein) and Glyma.13G068500 (cytochrome P450), were screened out for their different expressions between population parents at different seed growth and developmental stages. Thus, these results could provide novel insights for the genetic basis of seed size and shape in soybean. [ABSTRACT FROM AUTHOR]

Soybean mosaic virus (SMV) is a serious disease in soybean production, and the cultivation of resistant variety is the irreplaceable strategy to decrease the virus damage. However, the progress of SMV molecular breeding is limited partly due to the diverse types of SMV strains, solely resistance to virus strain of variety and minor genetic effects of QTLs. In the present study, one major pleiotropic additive QTL, qSMV‐D1b, with phenotypic variation explained that 27.04%–54.23% was identified on chromosome 2, and the favourable allele was from the resistant parent "Qihuang30". Meanwhile, the allele analysis of the flanking markers showed that the favourable genotypes presented high resistance to SC3 and SC7 strains with lower disease index, while the unfavourable genotypes presented high sensitivity with higher disease index. Furthermore, six candidate genes, including four novel resistance genes, Glyma.02G121000, Glyma.02G123000, Glyma.02G123200 and Glyma.02G124700 with different expression levels between resistant and sensitive parents were discovered through transcriptome sequencing and confirmed by quantitative real‐time PCR. The resistance genes and the tightly linked molecular markers can be used for SMV breeding in soybean. [ABSTRACT FROM AUTHOR]

Soybean provides irreplaceable protein and oil. Pods and seeds are essential soybean harvested organs. To illustrate the genetic basis of pod‐ and seed‐related traits, a natural population was assessed for eight related traits under four environments. In all, 15 associated SNPs were identified across multiple environments or controlling various traits, and 11 were verified through BLUP. Two SNPs on chromosome 15 were associated with six traits across multiple environments. Two SNPs on chromosome 2 were also associated with six traits under multiple environments. Significant phenotype differences between favourable and unfavourable alleles/haplotypes under four environments were detected. Significant association of pod length SNP and large‐pod phenotype (pod length ≥4.5 cm) was detected. The genetic basis of elite germplasms was analysed, and 'Chuxiu' possessed all of the 15 favourable alleles with best phenotype. Some candidate genes participating in cell wall structural remodelling, gibberellin and ethylene pathways were selected with different expressions in pod developmental stages. These results provided novel insights into genetic basis and molecular markers/candidate genes for soybean fresh pod and seed genetic improvement. [ABSTRACT FROM AUTHOR]

Expansins comprise four subfamilies, α-expansin (EXPA), β-expansin (EXPB), expansin-like A (EXLA), and expansin-like B (EXLB), which are involved in the regulation of root development and growth under abiotic stress. To date, few EXLB genes have been shown to respond to low phosphorus (P) in plants. In this study, we identified an EXLB gene, GmEXLB1 , by analyzing the transcription profiles of GmEXLBs in soybean. Quantitative analysis showed that GmEXLB1 was expressed and induced in the lateral roots of soybean under low P conditions. The observation of β-glucuronidase staining in transgenic Arabidopsis suggested that GmEXLB1 might be associated with lateral root emergence. GmEXLB1 overexpression altered the root architecture of transgenic Arabidopsis by increasing the number and length of lateral roots and the length of primary roots under low P conditions. Additionally, the length of the elongation zone and the average cell length in the elongation zone were increased in transgenic Arabidopsis. Increases in biomass and P content suggested that GmEXLB1 overexpression enhanced P acquisition in Arabidopsis. Overall, we conclude that GmEXLB1 expression is induced in soybean under low P conditions, and the overexpression of GmEXLB1 improves P acquisition by regulating root elongation and architecture in Arabidopsis , which provides a possible direction for research of the function of this gene in soybean. [ABSTRACT FROM AUTHOR]

Soybean is an important crops in the world due to its high seed protein and oil contents, while the contents of seed protein and oil attributed to the quantitative traits which were controlled by multiple minor-genes. Though there were many related QTLs reported, most of them only focused on the single additive QTLs, and few of them payed attentions to the epistatic QTLs. In this paper, a soybean RIL population (ZL) was used to study the epistatic QTLs of seed protein and oil contents by analyzing ZL population using Illumina BARCSoySNP6K Beadchip and detecting the phenotypes under four different environments. The results showed that there were 48 pairs of epistatic QTLs associated with protein content and 55 pairs of epistatic QTLs related to oil content in soybean seeds. Furthermore, there were 19 pairs of co-located epistatic QTLs controlling the protein and oil contents simultaneously. Among these co-located epistatic QTLs, 12 pairs of them shared the same QTL mapping regions, 2 pairs of them shared one same marker and five pairs of them had the QTLs regions not above 5 cM genetic distance, which could be regarded as the same QTL. Meanwhile, the results also showed that these epistatic QTLs located on nineteen chromosomes of soybean except for the chromosome 11. Thus, this study identified many epistatic QTLs for soybean seed protein and oil contents, which not only could be used for soybean quality genetic improvements in future, but also offered some evidences for reveal the negative relationships between seed protein and oil contents in soybean. [ABSTRACT FROM AUTHOR]

Induction and secretion of acid phosphatases (APases) is considered to be an important strategy for improving plant growth under conditions of low inorganic phosphate (Pi). Purple acid phosphatases (PAPs), are an important class of plant APases that could be secreted into the rhizosphere to utilize organic phosphorus (Po) for plant growth and development. To date, only a few members of the PAP family have been identified in soybean. In this paper, we identified a secreted PAP in soybean, GmPAP14, and investigated its role in utilizing external phytate, the main form of organic phosphorus in the soil. An analysis of its expression and promoter showed that GmPAP14 was mainly expressed in the root and was strongly induced following Po treatment, during which its expression expanded from meristematic to maturation zones and root hairs. In vitro enzyme assays indicated that GmPAP14 had a relatively high phytase activity. Furthermore, GmPAP14 overexpression increased secreted APase activities and phytase activities, leading to the improved use of external plant phytate, higher phosphorus content, and increased shoot weight. Thus, these results confirmed that GmPAP14 is an important gene induced in response to Po, and that it predominantly participates in utilizing external Po to enhance plant growth and development. [ABSTRACT FROM AUTHOR]

Soybean is important throughout the world not only due to the high seed protein and oil but also owing to the seed isoflavone. To improve the isoflavone concentration in seeds, detecting and mining the stable and reliable quantitative trait loci (QTLs) and related genes in multiple environments and genetic backgrounds become more and more important. In view of this, a F recombinant inbred line (RIL) population of 345 lines derived from a cross between Zheng 92116 and Liaodou14 (ZL) was genotyped using 1739 polymorphic SNP and 127 SSR markers in this study and was phenotyped for individual and total seed isoflavone in four environments over 2 years. In total, 48 additive QTLs, which explained 3.00-29.83% of seed isoflavone variation, were identified. Of them, eight QTLs ( qDA1_1, qGA1_1, qTIA1_1, qDA1_2, qGA1_2, qTIA1_2, qDA1_3, qTIA1_3) with phenotypic variation explained (PVE) ranging from 14.09 to 28.59% for daidzin, genistin, and total isoflavone were located on the same region of linkage group (LG) A1. These QTLs were further verified in another RIL population derived from Zheng 92116 × Qihuang 30 (ZQ). Meanwhile, the other four overlapping QTLs on linkage group B1, which were associated with glycitin content ( qGLB1_1, qGLB1_2, qGLB1_3, qGLB1_4) and explained 16.52 to 29.83% of phenotypic variation, were also verified using the ZQ population. Moreover, the individuals with different genotypes at the common flanking SNP markers for these QTLs on LGs A1 and B1 in the two mapping populations showed significant different isoflavone content, which further validate the QTL mapping results. And also, some candidate genes might participate in the isoflavone biosynthesis processes were found in these stable QTL regions. Thus, the novel and stable QTLs identified and verified in this study could be applied in marker-assisted selection breeding or map-based candidate genes cloning in soybean seed isoflavone genetic improvement in future. [ABSTRACT FROM AUTHOR]

Key message: GmPAP4 , a novel plant PAP gene in soybean, has phytase activity. Over-expressing GmPAP4 can enhance Arabidopsis growth when phytate is the sole P source in culture. Abstract: Phosphorus (P) is an important macronutrient for plant growth and development. However, most of the total P in soils is fixed into organic phosphate (Po). Purple acid phosphatase (PAP) can hydrolyze Po in the soil to liberate inorganic phosphate and enhance plant P utilization. We isolated a novel PAP gene, GmPAP4, from soybean ( Glycine max). It had an open reading frame of 1,329 bp, encoding 442 amino acid residues. Sequence alignment and phylogenetics analysis indicated that GmPAP4 was similar to other plant PAPs with large molecular masses. Quantitative real-time PCR analysis showed that the induced expression of GmPAP4 was greater in P-efficient genotype Zhonghuang15 (ZH15) than in P-inefficient genotype Niumaohuang (NMH) during the periods of flowering (28-35 days post phytate stress; DPP) and pod formation (49-63 DPP). Moreover, peak expression, at 63 DPP, was about 3-fold higher in 'ZH15' than in 'NMH'. Sub-cellular localization showed that GmPAP4 might be on plasma membrane or in cytoplasm. Over-expressing GmPAP4 in Arabidopsis resulted in significant rises in P acquisition and utilization compared with the wild-type (WT). Under phytate condition, transgenic Arabidopsis plants showed increases of approximately 132.7 % in dry weight and 162.6 % in shoot P content compared with the WT. Furthermore, when phytate was added as the sole P source in cultures, the activity of acid phosphatase was significantly higher in transgenic plants. Therefore, GmPAP4 is a novel PAP gene that functions in plant's utilization of organic phosphate especially under phytate condition. [ABSTRACT FROM AUTHOR]

Soybean is important throughout the world due to its high seed protein and oil, while the quality and quantity of seed amino acids need to be improved. To improve the multiple amino acid concentrations in soybean simultaneously, detecting and utilizing the pleiotropic quantitative trait loci (QTL) and related genes become increasingly important. In view of this, a F6:7 recombinant inbred line population was genotyped using 1739 polymorphic SNP and 127 SSR markers in the present study and was phenotyped for seventeen types of amino acids simultaneously. In total, twelve co-located or overlapped pleiotropic additive QTL clusters, which explained 2.38-16.79% of the amino acid variation, were identified. Of them, one novel pleiotropic QTL cluster with a phenotypic variation explained ranging from 8.84 to 16.79% for ten kinds of amino acid contents (glycine, alanine, isoleucine, leucine, valine, methionine, aspartic acid, glutamic acid, lysine and phenylalanine), was located at the same position on linkage group D2, and the confidence interval was only 0.8 cM. Moreover, the individuals in this family-based population (345 lines) and another cultivar-based population (254 varieties) with different genotypes at the common flanking markers for this QTL cluster showed significantly different amino acid contents, which further validated the QTL mapping results. Additionally, some candidate genes that might participate in the amino acid biosynthesis process were found in these pleiotropic QTL regions. Thus, novel pleiotropic QTL clusters could be applied in marker-assisted selection breeding or map-based candidate gene cloning in soybean for multiple amino acid genetic improvements in seed in the future. [ABSTRACT FROM AUTHOR]