Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid profile following seed germination.

Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource because of the presence of toxic gossypol. RNAi-knockdown of δ-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed (ULGCS). In the original study, we observed that RNAi plants, a month or older, maintain normal complement of gossypol and related terpenoids in the roots, foliage, floral organs, and young bolls. However, the terpenoid levels and profile of the RNAi lines during the early stages of germination, under normal conditions and in response to pathogen exposure, had not been examined. Results obtained in this study show that during the early stages of seed germination/seedling growth, in both non-transgenic and RNAi lines, the tissues derived directly from bulk of the seed kernel (cotyledon and hypocotyl) synthesize little, if any new terpenoids. However, the growing root tissue and the emerging true leaves of RNAi seedlings showed normal, wild-type terpenoid levels. Biochemical and molecular analyses showed that pathogen-challenged parts of RNAi seedlings are capable of launching a terpenoid-based defence response. Nine different RNAi lines were monitored for five generations. The results show that, unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability.

[1]  Allan G Green,et al.  High-Stearic and High-Oleic Cottonseed Oils Produced by Hairpin RNA-Mediated Post-Transcriptional Gene Silencing1 , 2002, Plant Physiology.

[2]  N. Powell.  Disease Complexes in Tobacco Involving Meloidogyne incognita and Certain Soil-Borne Fungi , 1971 .

[3]  D. Bassham,et al.  AtVPS45 Is a Positive Regulator of the SYP41/SYP61/VTI12 SNARE Complex Involved in Trafficking of Vacuolar Cargo1[OA] , 2009, Plant Physiology.

[4]  W. Liang,et al.  Expression pattern of (+)-δ-cadinene synthase genes and biosynthesis of sesquiterpene aldehydes in plants of Gossypium arboreum L. , 2000, Planta.

[5]  L. Hanson,et al.  Induction of Terpenoid Synthesis in Cotton Roots and Control of Rhizoctonia solani by Seed Treatment with Trichoderma virens. , 2000, Phytopathology.

[6]  X. Chen,et al.  Molecular cloning and functional identification of (+)-delta-cadinene-8-hydroxylase, a cytochrome P450 mono-oxygenase (CYP706B1) of cotton sesquiterpene biosynthesis. , 2001, The Plant journal : for cell and molecular biology.

[7]  S. Kennedy,et al.  FtsK-Dependent Dimer Resolution on Multiple Chromosomes in the Pathogen Vibrio cholerae , 2008, PLoS genetics.

[8]  S. Strauss,et al.  Efficient and stable transgene suppression via RNAi in field-grown poplars , 2008, Transgenic Research.

[9]  J. Connell,et al.  A comprehensive study of the use of a homologous promoter in antisense cotton lines exhibiting a high seed oleic acid phenotype. , 2005, Plant biotechnology journal.

[10]  M. Albrechtsen,et al.  Stability of RNA silencing-based traits in potato after virus infection , 2007, Molecular Breeding.

[11]  C. Bowler,et al.  Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes , 2005, Nature Biotechnology.

[12]  R. Stipanovic,et al.  Cotton Pest Resistance: The Role of Pigment Gland Constituents , 1999 .

[13]  Vinod Kumar,et al.  Defense-related gene expression and enzyme activities in transgenic cotton plants expressing an endochitinase gene from Trichoderma virens in response to interaction with Rhizoctonia solani , 2009, Planta.

[14]  Yoshikazu Tanaka,et al.  Flower color modification of Petunia hybrida commercial varieties by metabolic engineering , 2004 .

[15]  M. Essenberg,et al.  (+)-δ-Cadinene is a product of sesquiterpene cyclase activity in cotton , 1995 .

[16]  R. Mitchell,et al.  Loss of decreased-rubisco phenotype between generations of wheat transformed with antisense and sense rbcS , 2004 .

[17]  B. Keller,et al.  RNA Interference-Based Gene Silencing as an Efficient Tool for Functional Genomics in Hexaploid Bread Wheat1[W][OA] , 2006, Plant Physiology.

[18]  R. Stipanovic,et al.  Induction of delta-cadinene synthase and sesquiterpenoid phytoalexins in cotton by Verticillium dahliae. , 1999, Journal of agricultural and food chemistry.

[19]  M. Pierce,et al.  The phytoalexins desoxyhemigossypol and hemigossypol are elicited by Xanthomonas in Gossypium cotyledons , 1999 .

[20]  T. Nishio,et al.  Low glutelin content1: A Dominant Mutation That Suppresses the Glutelin Multigene Family via RNA Silencing in Rice Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.011452. , 2003, The Plant Cell Online.

[21]  Yoshikazu Tanaka,et al.  Plant Biotechnology 23, 123–127 (2006) , 2006 .

[22]  Christopher P. Bonin,et al.  High-lysine corn generated by endosperm-specific suppression of lysine catabolism using RNAi. , 2007, Plant biotechnology journal.

[23]  H. Vaucheret,et al.  SINE RNA Induces Severe Developmental Defects in Arabidopsis thaliana and Interacts with HYL1 (DRB1), a Key Member of the DCL1 Complex , 2008, PLoS genetics.

[24]  D. Llewellyn,et al.  Antisense Suppression of a (+)-δ-Cadinene Synthase Gene in Cotton Prevents the Induction of This Defense Response Gene during Bacterial Blight Infection But Not Its Constitutive Expression1[w] , 2005, Plant Physiology.

[25]  R. Stipanovic,et al.  Terpenoid aldehydes in upland cottons: analysis by aniline and HPLC methods , 1988 .

[26]  Qing Liu,et al.  hpRNA-Mediated Targeting of the Arabidopsis FAD2 Gene Gives Highly Efficient and Stable Silencing , 2002, Plant Physiology.

[27]  J. Carrington,et al.  Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. , 2001, Plant physiology.

[28]  R. Stipanovic,et al.  Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol , 2006, Proceedings of the National Academy of Sciences.

[29]  L. Puckhaber,et al.  Terpenoid aldehyde formation and lysigenous gland storage sites in cotton: variant with mature glands but suppressed levels of terpenoid aldehydes. , 2004, Phytochemistry.

[30]  C. W. Smith,et al.  Cotton α-Globulin Promoter: Isolation and Functional Characterization in Transgenic Cotton, Arabidopsis, and Tobacco , 2002, Transgenic Research.