Integrated Genome-Scale Analysis Identifies Novel Genes and Networks Underlying Senescence in Maize[OPEN]

A systematic characterization of natural diversity, coexpression network analysis, and experimental validation for understanding the genetic architecture of senescence and stay-green in maize. Premature senescence in annual crops reduces yield, while delayed senescence, termed stay-green, imposes positive and negative impacts on yield and nutrition quality. Despite its importance, scant information is available on the genetic architecture of senescence in maize (Zea mays) and other cereals. We combined a systematic characterization of natural diversity for senescence in maize and coexpression networks derived from transcriptome analysis of normally senescing and stay-green lines. Sixty-four candidate genes were identified by genome-wide association study (GWAS), and 14 of these genes are supported by additional evidence for involvement in senescence-related processes including proteolysis, sugar transport and signaling, and sink activity. Eight of the GWAS candidates, independently supported by a coexpression network underlying stay-green, include a trehalose-6-phosphate synthase, a NAC transcription factor, and two xylan biosynthetic enzymes. Source–sink communication and the activity of cell walls as a secondary sink emerge as key determinants of stay-green. Mutant analysis supports the role of a candidate encoding Cys protease in stay-green in Arabidopsis (Arabidopsis thaliana), and analysis of natural alleles suggests a similar role in maize. This study provides a foundation for enhanced understanding and manipulation of senescence for increasing carbon yield, nutritional quality, and stress tolerance of maize and other cereals.

[1]  Peng Liu,et al.  The Histone H3K4 Demethylase JMJ16 Represses Leaf Senescence in Arabidopsis[OPEN] , 2019, Plant Cell.

[2]  Yan Xia,et al.  Sweet Sorghum Originated through Selection of Dry, a Plant-Specific NAC Transcription Factor Gene[OPEN] , 2018, Plant Cell.

[3]  J. Doll,et al.  A guideline for leaf senescence analyses: from quantification to physiological and molecular investigations , 2018, Journal of experimental botany.

[4]  M. Paul,et al.  Trehalose 6-Phosphate Regulates Photosynthesis and Assimilate Partitioning in Reproductive Tissue , 2018, Plant Physiology.

[5]  Peter J. Bradbury,et al.  Novel Loci Underlie Natural Variation in Vitamin E Levels in Maize Grain[OPEN] , 2017, Plant Cell.

[6]  A. Wingler Transitioning to the Next Phase: The Role of Sugar Signaling throughout the Plant Life Cycle[OPEN] , 2017, Plant Physiology.

[7]  Xin-Guang Zhu,et al.  Leaf Photosynthetic Parameters Related to Biomass Accumulation in a Global Rice Diversity Survey1[OPEN] , 2017, Plant Physiology.

[8]  Wei Zhang,et al.  Editing of Mitochondrial Transcripts nad3 and cox2 by Dek10 Is Essential for Mitochondrial Function and Maize Plant Development , 2017, Genetics.

[9]  Kevin L. Schneider,et al.  Improved maize reference genome with single-molecule technologies , 2017, Nature.

[10]  A. Millar,et al.  Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves , 2017, BMC Plant Biology.

[11]  Yan Xia,et al.  Transcriptome profiling of developmental leaf senescence in sorghum (Sorghum bicolor) , 2016, Plant Molecular Biology.

[12]  Matthew P. Reynolds,et al.  Modelling and genetic dissection of staygreen under heat stress , 2016, Theoretical and Applied Genetics.

[13]  J. Lunn,et al.  A Tale of Two Sugars: Trehalose 6-Phosphate and Sucrose1[OPEN] , 2016, Plant Physiology.

[14]  O. Distl,et al.  Identification of genetic variants associated with semen traits in warmblood stallions , 2016 .

[15]  M. Diaz-Mendoza,et al.  HvPap-1 C1A protease actively participates in barley proteolysis mediated by abiotic stresses. , 2016, Journal of experimental botany.

[16]  Peter J. Bradbury,et al.  Identification of genetic variants associated with maize flowering time using an extremely large multi-genetic background population. , 2016, The Plant journal : for cell and molecular biology.

[17]  B. Dilkes,et al.  The Interaction of Genotype and Environment Determines Variation in the Maize Kernel Ionome , 2016, G3: Genes, Genomes, Genetics.

[18]  Sudhir Kumar,et al.  MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. , 2016, Molecular biology and evolution.

[19]  Xuecai Zhang,et al.  Identification of QTL for Early Vigor and Stay-Green Conferring Tolerance to Drought in Two Connected Advanced Backcross Populations in Tropical Maize (Zea mays L.) , 2016, PloS one.

[20]  Jin Ok Yang,et al.  Programming of Plant Leaf Senescence with Temporal and Inter-Organellar Coordination of Transcriptome in Arabidopsis1[OPEN] , 2016, Plant Physiology.

[21]  G. An,et al.  A Simple DNA Preparation Method for High Quality Polymerase Chain Reaction in Rice , 2016 .

[22]  Lisa C. Harper,et al.  MaizeGDB update: new tools, data and interface for the maize model organism database , 2015, Nucleic Acids Res..

[23]  Jos H. M. Schippers Transcriptional networks in leaf senescence. , 2015, Current opinion in plant biology.

[24]  M. Knight,et al.  Transcriptomic analysis comparing stay-green and senescent Sorghum bicolor lines identifies a role for proline biosynthesis in the stay-green trait , 2015, Journal of experimental botany.

[25]  L. M. Lagrimini,et al.  Expression of trehalose-6-phosphate phosphatase in maize ears improves yield in well-watered and drought conditions , 2015, Nature Biotechnology.

[26]  K. Moore,et al.  The impact of the HIRA histone chaperone upon global nucleosome architecture , 2015, Cell cycle.

[27]  C. Pieterse,et al.  β-Glucosidase BGLU42 is a MYB72-dependent key regulator of rhizobacteria-induced systemic resistance and modulates iron deficiency responses in Arabidopsis roots. , 2014, The New phytologist.

[28]  B. Biswal,et al.  Dehydration induced loss of photosynthesis in Arabidopsis leaves during senescence is accompanied by the reversible enhancement in the activity of cell wall β-glucosidase. , 2014, Journal of photochemistry and photobiology. B, Biology.

[29]  H. Thomas,et al.  The stay-green trait. , 2014, Journal of experimental botany.

[30]  Yana Zhu,et al.  OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice , 2014, Proceedings of the National Academy of Sciences.

[31]  J. Jeon,et al.  Recombinant Expression and Characterization of the Cytoplasmic Rice β-Glucosidase Os1BGlu4 , 2014, PloS one.

[32]  Xianghua Li,et al.  Overexpression of OsSWEET5 in Rice Causes Growth Retardation and Precocious Senescence , 2014, PloS one.

[33]  Jing Wang,et al.  CrossMap: a versatile tool for coordinate conversion between genome assemblies , 2014, Bioinform..

[34]  C. Masclaux-Daubresse,et al.  QTL meta-analysis in Arabidopsis reveals an interaction between leaf senescence and resource allocation to seeds , 2014, Journal of experimental botany.

[35]  Björn Usadel,et al.  Trimmomatic: a flexible trimmer for Illumina sequence data , 2014, Bioinform..

[36]  T. M. M. Câmara,et al.  Inheritance of the stay-green trait in tropical maize , 2014, Euphytica.

[37]  M. A. Pedraza,et al.  Insights into the Maize Pan-Genome and Pan-Transcriptome[W][OPEN] , 2014, Plant Cell.

[38]  A. Lusis,et al.  Systems genetics approaches to understand complex traits , 2013, Nature Reviews Genetics.

[39]  Langtao Xiao,et al.  Constitutive expression of cell wall invertase genes increases grain yield and starch content in maize. , 2013, Plant biotechnology journal.

[40]  Hong Gil Nam,et al.  Plant leaf senescence and death – regulation by multiple layers of control and implications for aging in general , 2013, Journal of Cell Science.

[41]  Zhonghai Li,et al.  LSD 2.0: an update of the leaf senescence database , 2013, Nucleic Acids Res..

[42]  Robert J. Elshire,et al.  Comprehensive genotyping of the USA national maize inbred seed bank , 2013, Genome Biology.

[43]  David M Brown,et al.  GUX1 and GUX2 glucuronyltransferases decorate distinct domains of glucuronoxylan with different substitution patterns. , 2013, The Plant journal : for cell and molecular biology.

[44]  C. Robin Buell,et al.  Maize Gene Atlas Developed by RNA Sequencing and Comparative Evaluation of Transcriptomes Based on RNA Sequencing and Microarrays , 2013, PloS one.

[45]  L. Emebiri QTL dissection of the loss of green colour during post-anthesis grain maturation in two-rowed barley , 2013, Theoretical and Applied Genetics.

[46]  I. Ciampitti,et al.  Grain Nitrogen Source Changes over Time in Maize: A Review , 2013 .

[47]  H. Thomas Senescence, ageing and death of the whole plant. , 2013, The New phytologist.

[48]  K. Krupinska,et al.  Plant senescence and crop productivity , 2013, Plant Molecular Biology.

[49]  Xiaohong Yang,et al.  Genome-wide association study dissects the genetic architecture of oil biosynthesis in maize kernels , 2012, Nature Genetics.

[50]  Meng Li,et al.  Genetics and population analysis Advance Access publication July 13, 2012 , 2012 .

[51]  Thomas L. Slewinski,et al.  Tie-dyed2 Encodes a Callose Synthase That Functions in Vein Development and Affects Symplastic Trafficking within the Phloem of Maize Leaves12[C][W][OA] , 2012, Plant Physiology.

[52]  R. Sekhon,et al.  Transcriptional and Metabolic Analysis of Senescence Induced by Preventing Pollination in Maize1[W][OA] , 2012, Plant Physiology.

[53]  C. Funk,et al.  Senescence-associated proteases in plants. , 2012, Physiologia plantarum.

[54]  Pablo Cingolani,et al.  © 2012 Landes Bioscience. Do not distribute. , 2022 .

[55]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[56]  Chunqing Zhang,et al.  QTL mapping for stay-green in maize (Zea mays) , 2012, Canadian Journal of Plant Science.

[57]  David R. Kelley,et al.  Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks , 2012, Nature Protocols.

[58]  S. Gan,et al.  Convergence and divergence in gene expression profiles induced by leaf senescence and 27 senescence-promoting hormonal, pathological and environmental stress treatments. , 2012, Plant, cell & environment.

[59]  M. Paul,et al.  Trehalose 6-Phosphate Is Required for the Onset of Leaf Senescence Associated with High Carbon Availability1[W][OA] , 2012, Plant Physiology.

[60]  A. Myburg,et al.  SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis fibres and increases fibre cell area in Eucalyptus , 2011, BMC Plant Biology.

[61]  A. Wingler Interactions between flowering and senescence regulation and the influence of low temperature in Arabidopsis and crop plants , 2011 .

[62]  Syed Haider,et al.  Ensembl BioMarts: a hub for data retrieval across taxonomic space , 2011, Database J. Biol. Databases Curation.

[63]  V. Allard,et al.  Anthesis date mainly explained correlations between post-anthesis leaf senescence, grain yield, and grain protein concentration in a winter wheat population segregating for flowering time QTLs. , 2011, Journal of experimental botany.

[64]  C. Wilkerson,et al.  The DUF579 domain containing proteins IRX15 and IRX15-L affect xylan synthesis in Arabidopsis. , 2011, The Plant journal : for cell and molecular biology.

[65]  Wei Wang,et al.  [A cloud detection algorithm for MODIS images combining Kmeans clustering and multi-spectral threshold method]. , 2011, Guang pu xue yu guang pu fen xi = Guang pu.

[66]  M. Carena,et al.  Area Under the Dry Down Curve (AUDDC): A Method to Evaluate Rate of Dry Down in Maize , 2010 .

[67]  J. Boyer,et al.  Sugar input, metabolism, and signaling mediated by invertase: roles in development, yield potential, and response to drought and heat. , 2010, Molecular plant.

[68]  P. Starks,et al.  Rapid analysis of nonstructural carbohydrate components in grass forage using microplate enzymatic assays. , 2010 .

[69]  Donald N. Duvick,et al.  Long‐Term Selection in a Commercial Hybrid Maize Breeding Program , 2010 .

[70]  F. Rolland,et al.  Sugar signals and molecular networks controlling plant growth. , 2010, Current opinion in plant biology.

[71]  Matthew D. Young,et al.  Gene ontology analysis for RNA-seq: accounting for selection bias , 2010, Genome Biology.

[72]  Trevor Hastie,et al.  Regularization Paths for Generalized Linear Models via Coordinate Descent. , 2010, Journal of statistical software.

[73]  Guihua Bai,et al.  Modeling and mapping QTL for senescence-related traits in winter wheat under high temperature , 2010, Molecular Breeding.

[74]  Davis J. McCarthy,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[75]  J. Carbonell,et al.  Antagonistic interactions between Arabidopsis K-homology domain genes uncover PEPPER as a positive regulator of the central floral repressor FLOWERING LOCUS C. , 2009, Developmental biology.

[76]  Steven J. M. Jones,et al.  Circos: an information aesthetic for comparative genomics. , 2009, Genome research.

[77]  A. Fischer,et al.  Sugars, senescence, and ageing in plants and heterotrophic organisms. , 2009, Journal of experimental botany.

[78]  S. Rothstein,et al.  Over-expression of STP13, a hexose transporter, improves plant growth and nitrogen use in Arabidopsis thaliana seedlings. , 2009, Plant, cell & environment.

[79]  P. J. Andralojc,et al.  Inhibition of SNF1-Related Protein Kinase1 Activity and Regulation of Metabolic Pathways by Trehalose-6-Phosphate1[W][OA] , 2009, Plant Physiology.

[80]  S. Dong,et al.  QTL mapping of maize (Zea mays) stay-green traits and their relationship to yield. , 2009 .

[81]  R. Meeley,et al.  Sucrose transporter1 functions in phloem loading in maize leaves , 2009 .

[82]  T. Roitsch,et al.  Metabolic regulation of leaf senescence: interactions of sugar signalling with biotic and abiotic stress responses. , 2008, Plant biology.

[83]  M. Sagi,et al.  A critical role for ureides in dark and senescence-induced purine remobilization is unmasked in the Atxdh1 Arabidopsis mutant. , 2008, The Plant journal : for cell and molecular biology.

[84]  Matthijs Tollenaar,et al.  Physiological Basis of Successful Breeding Strategies for Maize Grain Yield , 2007 .

[85]  Honglin Xu,et al.  Molecular cloning and expression analysis of a monosaccharide transporter gene OsMST4 from rice (Oryza sativa L.) , 2007, Plant Molecular Biology.

[86]  H. Paik,et al.  Quantitative trait loci associated with functional stay-green SNU-SG1 in rice. , 2007, Molecules and cells.

[87]  Feng Luo,et al.  Constructing gene co-expression networks and predicting functions of unknown genes by random matrix theory , 2007, BMC Bioinformatics.

[88]  D. Braun,et al.  tie-dyed1 Functions Non-Cell Autonomously to Control Carbohydrate Accumulation in Maize Leaves1[C][W][OA] , 2007, Plant Physiology.

[89]  V. Grbić,et al.  Vacuolar cysteine proteases of wheat (Triticum aestivum L.) are common to leaf senescence induced by different factors. , 2007, Journal of experimental botany.

[90]  H. Nguyen,et al.  Sorghum stay-green QTL individually reduce post-flowering drought-induced leaf senescence. , 2006, Journal of experimental botany.

[91]  E. Baena-González,et al.  Sugar sensing and signaling in plants: conserved and novel mechanisms. , 2006, Annual review of plant biology.

[92]  Morten H. H. Nørholm,et al.  Expression of the Arabidopsis high‐affinity hexose transporter STP13 correlates with programmed cell death , 2006, FEBS letters.

[93]  M. McMullen,et al.  A unified mixed-model method for association mapping that accounts for multiple levels of relatedness , 2006, Nature Genetics.

[94]  Jian Wang,et al.  A microarray analysis of the rice transcriptome and its comparison to Arabidopsis. , 2005, Genome research.

[95]  R. Shishido,et al.  QTL-based analysis of leaf senescence in an indica/japonica hybrid in rice (Oryza sativa L.) , 2005, Theoretical and Applied Genetics.

[96]  Stephen H. Bryant,et al.  CD-Search: protein domain annotations on the fly , 2004, Nucleic Acids Res..

[97]  M. Bogyo,et al.  Activity Profiling of Papain-Like Cysteine Proteases in Plants1 , 2004, Plant Physiology.

[98]  S. Gan,et al.  Transcriptome of Arabidopsis leaf senescence , 2004 .

[99]  T. Roitsch,et al.  Extracellular Invertase Is an Essential Component of Cytokinin-Mediated Delay of Senescence , 2004, The Plant Cell Online.

[100]  A. Wingler,et al.  Spatial patterns and metabolic regulation of photosynthetic parameters during leaf senescence. , 2004, The New phytologist.

[101]  Q. Zhang,et al.  The genetic basis of stay-green in rice analyzed in a population of doubled haploid lines derived from an indica by japonica cross , 2004, Theoretical and Applied Genetics.

[102]  E. Harrison,et al.  The molecular analysis of leaf senescence--a genomics approach. , 2002, Plant biotechnology journal.

[103]  J. Chory,et al.  BES1 Accumulates in the Nucleus in Response to Brassinosteroids to Regulate Gene Expression and Promote Stem Elongation , 2002, Cell.

[104]  H. Rogers,et al.  Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals. , 2002, Journal of experimental botany.

[105]  E. B. Dumbroff,et al.  Ultrastructural and biochemical changes in chloroplasts during Brassica napus senescence , 2001 .

[106]  B. Quirino,et al.  One of two tandem Arabidopsis genes homologous to monosaccharide transporters is senescence-associated , 2001, Plant Molecular Biology.

[107]  Matthijs Tollenaar,et al.  Response of maize leaf photosynthesis to low temperature during the grain-filling period , 2000 .

[108]  R. Amasino,et al.  Molecular aspects of leaf senescence. , 2000, Trends in plant science.

[109]  O. Crasta,et al.  Molecular mapping of QTLs conferring stay-green in grain sorghum (Sorghum bicolor L. Moench). , 2000, Genome.

[110]  Sjef Smeekens,et al.  SUGAR-INDUCED SIGNAL TRANSDUCTION IN PLANTS. , 2000, Annual review of plant physiology and plant molecular biology.

[111]  K Maxwell,et al.  Chlorophyll fluorescence--a practical guide. , 2000, Journal of experimental botany.

[112]  J. Prioul,et al.  Characterization of two members of the maize gene family, Incw3 and Incw4, encoding cell-wall invertases. , 2000, Gene.

[113]  H. Thomas,et al.  Five ways to stay green. , 2000, Journal of experimental botany.

[114]  D. Luthe,et al.  Characterization of three distinct cDNA clones encoding cysteine proteinases from maize (Zea mays L.) callus , 1999, Plant Molecular Biology.

[115]  M. Tollenaar,et al.  Source : sink ratio and leaf senescence in maize:: II. Nitrogen metabolism during grain filling , 1999 .

[116]  Jianhua Zhang,et al.  Modifications in photosystem II photochemistry in senescent leaves of maize plants , 1998 .

[117]  P. Goldsbrough,et al.  Genetic analysis of post-flowering drought tolerance and components of grain development in Sorghum bicolor (L.) Moench , 1997, Molecular Breeding.

[118]  T. Roitsch,et al.  Co-ordinated induction of mRNAs for extracellular invertase and a glucose transporter in Chenopodium rubrum by cytokinins. , 1997, The Plant journal : for cell and molecular biology.

[119]  S. Clouse Molecular genetic studies confirm the role of brassinosteroids in plant growth and development. , 1996, The Plant journal : for cell and molecular biology.

[120]  L. D. Vré,et al.  Ethylene‐insensitive floral senescence in Sandersonia aurantiaca (Hook.) , 1995 .

[121]  E. Lander,et al.  Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results , 1995, Nature Genetics.

[122]  C. Rivin,et al.  Sequence and Regulation of a Late Embryogenesis Abundant Group 3 Protein of Maize , 1995, Plant physiology.

[123]  J. Chory,et al.  A Role for Cytokinins in De-Etiolation in Arabidopsis (det Mutants Have an Altered Response to Cytokinins) , 1994, Plant physiology.

[124]  K. Shinozaki,et al.  Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana. , 1993, Gene.

[125]  C. Boyer,et al.  Alterations in Carbohydrate Intermediates in the Endosperm of Starch-Deficient Maize (Zea mays L.) Genotypes. , 1992, Plant physiology.

[126]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[127]  F. Salamini,et al.  A Major Gene for Delayed Senescence in Maize. Pattern of Photosynthates Accumulation and Inheritance , 1986 .

[128]  J. Felsenstein CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP , 1985, Evolution; international journal of organic evolution.

[129]  F. Below,et al.  Differential Senescence of Maize Hybrids following Ear Removal : II. Selected Leaf. , 1984, Plant physiology.

[130]  R. Creelman,et al.  Effect of Senescene and Nonsenescence on Carbohydrates in Sorghum During Late Kernel Maturity States1 , 1983 .

[131]  F. Below,et al.  Interaction of carbon and nitrogen metabolism in the productivity of maize. , 1982, Plant physiology.

[132]  F. Below,et al.  Availability of reduced N and carbohydrates for ear development of maize. , 1981, Plant physiology.

[133]  Jingchu Luo,et al.  Construction of the Leaf Senescence Database and Functional Assessment of Senescence-Associated Genes. , 2017, Methods in molecular biology.

[134]  Paul Theodor Pyl,et al.  HTSeq – A Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[135]  Ronan C O'Malley,et al.  A user's guide to the Arabidopsis T-DNA insertion mutant collections. , 2015, Methods in molecular biology.

[136]  Liyan,et al.  QTL mapping for stay-green in maize (Zea mays) , 2012 .

[137]  Michael Popelka,et al.  Genetic architecture of stay-green in maize , 2012 .

[138]  Stacey S. Cherny,et al.  Evaluating the effective numbers of independent tests and significant p-value thresholds in commercial genotyping arrays and public imputation reference datasets , 2011, Human Genetics.

[139]  Christopher A. Penfold,et al.  High-Resolution Temporal Profiling of Transcripts during Arabidopsis Leaf Senescence Reveals a Distinct Chronology of Processes and Regulation , 2011 .

[140]  C. Masclaux-Daubresse,et al.  QTL analysis for sugar-regulated leaf senescence supports flowering-dependent and -independent senescence pathways. , 2010, The New phytologist.

[141]  P. Holm,et al.  Transcriptome analysis of senescence in the flag leaf of wheat (Triticum aestivum L.). , 2007, Plant biotechnology journal.

[142]  H. Nam,et al.  Leaf senescence. , 2007, Annual review of plant biology.

[143]  A. Wingler,et al.  The role of sugars in integrating environmental signals during the regulation of leaf senescence. , 2006, Journal of experimental botany.

[144]  Alan M. Jones,et al.  The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis. , 2004, Phytochemistry.

[145]  Govindjee,et al.  Chlorophyll a Fluorescence , 2004, Advances in Photosynthesis and Respiration.

[146]  Keith R. Davis,et al.  Growth Stage–Based Phenotypic Analysis of Arabidopsis: A Model for High Throughput Functional Genomics in Plants , 2001 .

[147]  R. Tibshirani Regression Shrinkage and Selection via the Lasso , 1996 .

[148]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[149]  H. Lichtenthaler CHLOROPHYLL AND CAROTENOIDS: PIGMENTS OF PHOTOSYNTHETIC BIOMEMBRANES , 1987 .

[150]  L. Pauling,et al.  Evolutionary Divergence and Convergence in Proteins , 1965 .