A gene encoding an abscisic acid biosynthetic enzy

Authors: Jason Argyris María José Truco Oswaldo Ochoa Leah McHale Peetambar Dahal Allen Van Deynze Richard W Michelmore Kent J Bradford

Publish Date: 2010/08/12

Volume: 122, Issue: 1, Pages: 95-108

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Abstract

Thermoinhibition or failure of seeds to germinate when imbibed at warm temperatures can be a significant problem in lettuce Lactuca sativa L production The reliability of stand establishment would be improved by increasing the ability of lettuce seeds to germinate at high temperatures Genes encoding germination or dormancyrelated proteins were mapped in a recombinant inbred line population derived from a cross between L sativa cv Salinas and L serriola accession UC96US23 This revealed several candidate genes that are located in the genomic regions containing quantitative trait loci QTLs associated with temperature and light requirements for germination In particular LsNCED4 a temperatureregulated gene in the biosynthetic pathway for abscisic acid ABA a germination inhibitor mapped to the center of a previously detected QTL for high temperature germination Htg61 from UC96US23 Three sets of sister BC3S2 nearisogenic lines NILs that were homozygous for the UC96US23 allele of LsNCED4 at Htg61 were developed by backcrossing to cv Salinas and markerassisted selection followed by selfing The maximum temperature for germination of NIL seed lots with the UC96US23 allele at LsNCED4 was increased by 2–3°C when compared with sister NIL seed lots lacking the introgression In addition the expression of LsNCED4 was two to threefold lower in the former NIL lines as compared to expression in the latter Together these data strongly implicate LsNCED4 as the candidate gene responsible for the Htg61 phenotype and indicate that decreased ABA biosynthesis at high imbibition temperatures is a major factor responsible for the increased germination thermotolerance of UC96US23 seedsThe genus Lactuca L belongs to the Compositae or Asteraceae family and is represented by approximately 100 species distributed in warm and temperate regions of Europe Asia North America Africa and Australia Dziechciarkova et al 2004 Lebeda et al 2004 Lettuce Lactuca sativa L is an important crop species with an annual farmgate value of over 27 billion in the US in 2007 NASS 2008 Thermoinhibition inhibition of seed germination by high temperature in lettuce can delay or prevent germination in warm season plantings resulting in reduced field emergence and stand establishment Valdes et al 1985 Thermoinhibitory conditions often occur in major winter lettuce production areas of California and Arizona where temperatures permissive of germination are exceeded during late summer and fall plantings Delays in germination and asynchronous emergence can diminish yield Cantliffe et al 1981 necessitate multiple harvests and create other management problems through nonuniformity of crop development resulting in losses in quality and profitability Benjamin 1990The maximum temperatures for lettuce seed germination are genetically heritable Argyris et al 2005 Guzman et al 1992 and are influenced by environmental factors such as the temperature and light environment during seed maturation and germination Contreras et al 2008 2009 Gray et al 1988 Harrington and Thompson 1952 Hayashi et al 2008 Kozarewa et al 2006 Kristie and Fielding 1994 Saini et al 1989 Sung et al 1998 An intact endosperm envelope that encloses the lettuce embryo but not the outer fused testa and pericarp is essential for the imposition of thermoinhibition Borthwick and Robbins 1928 Dunlap et al 1990 Globerson et al 1974 Sung et al 2008Physiologically the maximum germination temperature of lettuce seeds appears to be determined primarily through the antagonistic interactions of gibberellins GA and abscisic acid ABA Gonai et al 2004 Toh et al 2008 and the regulation of their synthesis and degradation by phytochrome Sawada et al 2008a b Seo et al 2006 Seeds of ABAdeficient and insensitive mutants aba1 and abi3 in Arabidopsis thaliana germinate at supraoptimal temperatures supporting a major role for ABA in the thermoinhibition mechanism Tamura et al 2006 The expression of genes involved in ABA biosynthesis in Arabidopsis eg AtNCED genes encoding 9cisepoxycarotinoid dioxygenases is upregulated at high imbibition temperatures with AtNCED9 playing the major role in thermoinhibition while the expression of genes involved in GA synthesis eg AtGA3ox genes encoding GA 3βhydroxylases is downregulated Toh et al 2008 In cultivated lettuce ABA content is maintained at elevated levels when seeds are imbibed at high temperatures but decreases rapidly when imbibition occurs at optimal temperatures for germination Argyris et al 2008b Toh et al 2008 Yoshioka et al 1998 Gibberellins promote ABA catabolism and thus increase the maximum temperature of lettuce seed germination Gonai et al 2004 Ethylene application also promotes germination at high temperatures and endogenous ethylene production is greater in seeds capable of germinating at high temperature Kozarewa et al 2006 Prusinski and Khan 1990 Saini et al 1986 Large scale transcriptional profiling of genes involved in these hormonal biosynthesis and deactivation pathways showed that genetic variation in maximum germination temperature was associated with reciprocal changes in expression of ABA versus GA and ethylene biosynthesis metabolism and response genes Argyris et al 2008bAs in many other crop species molecular markers have been utilized in Lactuca species for taxonomic and biodiversity studies germplasm identification improving disease resistance dissecting quantitative traits and markerassisted selection MAS for crop improvement Argyris et al 2005 Dziechciarkova et al 2004 Grube et al 2005 Lebeda et al 2007 Michelmore et al 1991 Molecular marker identification has been enhanced through the development of an expressed sequence tag EST database of over 135000 ESTs from multiple Lactuca species through the Compositae Genome Project CGP http//cgpdbucdavisedu/ This database has aided the construction of an integrated genetic map using six inter and intraspecific crosses of Lactuca spp and comprises over 2700 markers spanning 1505 cM in nine linkage groups LGs corresponding to the nine chromosomes of Lactuca Truco et al 2007 More recently marker density of the integrated genetic map has been greatly augmented using a tiled microarray representing ~29000 unigenes for massively parallel genetic mapping allowing over 10000 polymorphic loci to be mapped van Leeuwen et al 2009 These resources have facilitated the identification of quantitative trait loci QTLs and the genetic mapping of candidate genes associated with seed germination dormancy and thermotolerance in a recombinant inbred line RIL population derived from L sativa cv Salinas and L serriola accession UC96US23 Argyris et al 2005 2008a For example a major QTL for high temperature germination Htg61 collocated with LsNCED4 a key regulated gene in the ABA biosynthetic pathway Argyris et al 2008b The expression of this gene and seed ABA content were inversely correlated with the maximum germination temperatureThe dissection of the genetic architecture of quantitative traits is accomplished effectively through backcrossing and the development of nearisogenic lines NILs A gene chromosomal segment or QTL can be isolated in a different and uniform genetic background that of the recurrent parent RP and its additive effects validated MacKay et al 2009 NILs have broad applications as immortal mapping populations with greater mapping power as compared to RILs Keurentjes et al 2007 In lettuce NILs and backcrossinbred line populations have been used to study herbicide resistance to detect markers associated with disease resistance and to examine potentially beneficial traits derived from wild germplasm Jeuken and Lindhout 2004 MallorySmith et al 1990 Paran et al 1991 NILs are particularly critical for studies of seed traits such as dormancy as the status of an individual seed is scored as either germinated or not qualitatively while populations of seeds exhibit quantitative variation in dormancy characteristics Bradford 1996 To this end NILs have been used effectively in validating seed dormancy QTLs in Arabidopsis and rice Oryza sativa L Bentsink et al 2006 Gu et al 2006 In lettuce the Htg61 QTL for high temperature germination conferred significant increases in the maximum germination temperature in advanced backcross BC progeny Argyris et al 2008a

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