Plant Cell Reports

, Volume 38, Issue 8, pp 965–980 | Cite as

Abscisic acid deficiency caused by phytoene desaturase silencing is associated with dwarfing syndrome in citrus

  • Nabil KillinyEmail author
  • Yasser Nehela
Original Article


Key message

In citrus, abscisic acid-deficiency was associated with a dwarfing phenotype, slow growth, small leaves, decreased fresh weight, and faster water loss. ABA supplementation reversed the dwarfing phenotype and enhanced growth.


Abscisic acid (ABA) is a ubiquitously distributed phytohormone, which is almost produced by all living kingdoms. In plants, ABA plays pleiotropic physiological roles in growth, development, and stress responses. We explored the hidden relationship between ABA deficiency, and citrus dwarfing. We used targeted-HPLC, targeted-GC–MS, molecular genetics, immunoassays, and gene expression techniques to investigate the effects of the silencing of phytoene desaturase (PDS) gene on the ABA-biosynthetic pathway, endogenous ABA content, and other phytohormones. Silencing of PDS directly suppressed the carotenoids compounds involved in ABA biosynthesis, altered phytohormonal profile, and caused phytoene accumulation and ABA deficiency. The reduction of ABA presumably due to the limited availability of its precursor, zeaxanthin. The ABA-deficient citrus cuttings displayed photobleaching, a dwarf phenotype with impaired growth characteristics that included slow growth, small leaves, decreased fresh weight, and faster water loss. ABA supplementation enhanced the growth and reversed the dwarfing phenotype of the ABA-deficient cuttings. Our data demonstrate that ABA-deficiency may lead to dwarfing phenotype and impaired growth in citrus cuttings. The negative influence of ABA-deficiency on growth rate is the result of altered water relations. Addition of ABA to the CTV-tPDS roots restored shoot growth and reversed the dwarfing phenotype.


Citrus sinensis Phytoene desaturase Abscisic acid Virus-induced gene silencing Dwarfing 



The authors acknowledge our CREC colleagues for their helpful discussion. We thank Dr. Faraj Hijaz and Shelley E. Jones for the technical assistance and Floyd Butz for maintaining the trees in greenhouses.

Author contribution statement

NK and YN conceptualized the research, performed experiments, conducted data analysis, visualized the data, and wrote the manuscript; NK administered the project.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.


  1. Addicott FT, Carns HR, Lyon JL et al (1964) On the physiology of abscisins. Régulateurs naturels de la croissance végétale, pp 687–703Google Scholar
  2. Addicott FT, Lyon JL, Ohkuma K et al (1968) Abscisic acid: a new name for abscisin II (Dormin). Science 159:1493CrossRefGoogle Scholar
  3. Agustí J, Zapater M, Iglesias DJ et al (2007) Differential expression of putative 9-cis-epoxycarotenoid dioxygenases and abscisic acid accumulation in water stressed vegetative and reproductive tissues of citrus. Plant Sci 172:85–94CrossRefGoogle Scholar
  4. Alférez F, Sala JM, Sanchez-Ballesta MT et al (2005) A comparative study of the postharvest performance of an ABA-deficient mutant of oranges: I. Physiological and quality aspects. Postharvest Biol Technol 37:222–231CrossRefGoogle Scholar
  5. Alquézar B, Zacarías L, Rodrigo MJ (2009) Molecular and functional characterization of a novel chromoplast-specific lycopene β-cyclase from Citrus and its relation to lycopene accumulation. J Exp Bot 60:1783–1797CrossRefGoogle Scholar
  6. Bari R, Jones JDG (2009) Role of plant hormones in plant defence responses. Plant Mol Biol 69:473–488CrossRefGoogle Scholar
  7. Barrero JM, Piqueras P, González-Guzmán M et al (2005) A mutational analysis of the ABA1 gene of Arabidopsis thaliana highlights the involvement of ABA in vegetative development. J Exp Bot 56:2071–2083CrossRefGoogle Scholar
  8. Becker A, Lange M (2010) VIGS—genomics goes functional. Trends Plant Sci 15:1–4CrossRefGoogle Scholar
  9. Bradford KJ (1983) Water relations and growth of the flacca tomato mutant in relation to abscisic acid. Plant Physiol 72:251–255CrossRefGoogle Scholar
  10. Butenko MA, Patterson SE, Grini PE et al (2003) Inflorescence deficient in abscission controls floral organ abscission in Arabidopsis and identifies a novel family of putative ligands in plants. Plant Cell 15:2296–2307CrossRefGoogle Scholar
  11. Chamovitz D, Sandmann G, Hirschberg J (1993) Molecular and biochemical characterization of herbicide-resistant mutants of cyanobacteria reveals that phytoene desaturation is a rate-limiting step in carotenoid biosynthesis. J Biol Chem 268:17348–17353Google Scholar
  12. Cheng W-H, Endo A, Zhou L et al (2002) A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions. Plant Cell 14:2723–2743CrossRefGoogle Scholar
  13. Cheng ZJ, Zhao XY, Shao XX et al (2014) Abscisic acid regulates early seed development in Arabidopsis by ABI5-mediated transcription of SHORT HYPOCOTYL UNDER BLUE1. Plant Cell 26:1053–1068CrossRefGoogle Scholar
  14. Cracker LE, Abeles FB (1969) Abscission: role of abscisic Acid. Plant Physiol 44:1144–1149CrossRefGoogle Scholar
  15. Cramer GR (2002) Response of abscisic acid mutants of Arabidopsis to salinity. Funct Plant Biol 29:561CrossRefGoogle Scholar
  16. Crocoll C, Kettner J, Dörffling K (1991) Abscisic acid in saprophytic and parasitic species of fungi. Phytochemistry 30:1059–1060CrossRefGoogle Scholar
  17. Domagalska MA, Sarnowska E, Nagy F, Davis SJ (2010) Genetic analyses of interactions among gibberellin, abscisic acid, and brassinosteroids in the control of flowering time in Arabidopsis thaliana. PLoS One 5:e14012CrossRefGoogle Scholar
  18. Dooner HK (1985) Viviparous-1 mutation in maize conditions pleiotropic enzyme deficiencies in the aleurone. Plant Physiol 77:486–488CrossRefGoogle Scholar
  19. Du H, Wu N, Chang Y et al (2013) Carotenoid deficiency impairs ABA and IAA biosynthesis and differentially affects drought and cold tolerance in rice. Plant Mol Biol 83:475–488CrossRefGoogle Scholar
  20. Eagles CF, Wareing PF (1963) Dormancy regulators in woody plants: experimental induction of dormancy in Betula pubescens. Nature 199:874–875CrossRefGoogle Scholar
  21. Eisenreich W, Bacher A, Arigoni D, Rohdich F (2004) Biosynthesis of isoprenoids via the non-mevalonate pathway. Cell Mol Life Sci 61:1401–1426CrossRefGoogle Scholar
  22. Finkelstein R (2013) Abscisic acid synthesis and response. Arab B/Am Soc Plant Biologists 11:e0166Google Scholar
  23. Forchetti G, Masciarelli O, Alemano S et al (2007) Endophytic bacteria in sunflower (Helianthus annuus L.): isolation, characterization, and production of jasmonates and abscisic acid in culture medium. Appl Microbiol Biotechnol 76:1145–1152CrossRefGoogle Scholar
  24. Fraser P, Bramley PM (2004) The biosynthesis and nutritional uses of carotenoids. Prog Lipid Res 43:228–265CrossRefGoogle Scholar
  25. Galpaz N, Wang Q, Menda N et al (2008) Abscisic acid deficiency in the tomato mutant high-pigment 3 leading to increased plastid number and higher fruit lycopene content. Plant J 53:717–730CrossRefGoogle Scholar
  26. Hajeri S, Killiny N, El-Mohtar C et al (2014) Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease (Huanglongbing). J Biotechnol 176:42–49CrossRefGoogle Scholar
  27. Hall HK, McWha JA (1982) Abscisic acid and wheat leaf senescence: the effect of pre-treating the intact plant. Zeitschrift für Pflanzenphysiologie 106:371–373CrossRefGoogle Scholar
  28. Hijaz F, Killiny N (2014) Collection and chemical composition of phloem sap from Citrus sinensis L. Osbeck (sweet orange). PLoS One 9:1–11CrossRefGoogle Scholar
  29. Inomata M, Hirai N, Yoshida R, Ohigashi H (2004a) Biosynthesis of abscisic acid by the direct pathway via ionylideneethane in a fungus, Cercospora cruenta. Biosci Biotechnol Biochem 68:2571–2580CrossRefGoogle Scholar
  30. Inomata M, Hirai N, Yoshida R, Ohigashi H (2004b) The biosynthetic pathway to abscisic acid via ionylideneethane in the fungus Botrytis cinerea. Phytochemistry 65:2667–2678CrossRefGoogle Scholar
  31. Izquierdo-Bueno I, González-Rodríguez VE, Simon A et al (2018) Biosynthesis of abscisic acid in fungi: identification of a sesquiterpene cyclase as the key enzyme in Botrytis cinerea. Environ Microbiol 20:2469–2482CrossRefGoogle Scholar
  32. Jaschke WD, Peuke AD, Pate JS, Hartung W (1997) Transport, synthesis and catabolism of abscisic acid (ABA) in intact plants of castor bean (Ricinus communis L.) under phosphate deficiency and moderate salinity. J Exp Bot 48:1737–1747CrossRefGoogle Scholar
  33. Killiny N, Nehela Y (2017) One target, two mechanisms: the impact of “Candidatus Liberibacter asiaticus” and its vector, Diaphorina citri, on citrus leaf pigments. Mol Plant Microbe Interact 30:543–556CrossRefGoogle Scholar
  34. Koornneef M, Jorna ML, Brinkhorst-van der Swan DLC, Karssen CM (1982) The isolation of abscisic acid (ABA) deficient mutants by selection of induced revertants in non-germinating gibberellin sensitive lines of Arabidopsis thaliana (L.) heynh. Theor Appl Genet 61:385–393CrossRefGoogle Scholar
  35. Kumagai MH, Donson J, Della-Cioppa G et al (1995) Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc Natl Acad Sci USA 92:1679–1683CrossRefGoogle Scholar
  36. Li H-H, Hao R-L, Wu S-S et al (2011) Occurrence, function and potential medicinal applications of the phytohormone abscisic acid in animals and humans. Biochem Pharmacol 82:701–712CrossRefGoogle Scholar
  37. Lichtenthaler HK (1999) The 1-deoxy-d-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 50:47–65CrossRefGoogle Scholar
  38. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the \( 2^{{ - \Delta \Delta C_{\text{T}} }} \) method. Methods 25:402–408CrossRefGoogle Scholar
  39. López-Ráez JA, Shirasu K, Foo E (2017) Strigolactones in plant interactions with beneficial and detrimental organisms: the Yin and Yang. Trends Plant Sci 22:527–537CrossRefGoogle Scholar
  40. Mafra V, Kubo KS, Alves-Ferreira M et al (2012) Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions. PLoS One 7:e31263CrossRefGoogle Scholar
  41. Marin E, Nussaume L, Quesada A et al (1996) Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana. EMBO J 15:2331–2342CrossRefGoogle Scholar
  42. Maršálek B, Zahradníčková H, Hronková M (1992) Extracellular abscisic acid produced by cyanobacteria under salt stress. J Plant Physiol 139:506–508CrossRefGoogle Scholar
  43. McCarty DR, Carson CB, Stinard PS, Robertson DS (1989) Molecular analysis of viviparous-1: an abscisic acid-insensitive mutant of maize. Plant Cell 1:523–532CrossRefGoogle Scholar
  44. Milborrow BV (2001) The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis. J Exp Bot 52:1145–1164CrossRefGoogle Scholar
  45. Motomitsu A, Sawa S, Ishida T (2015) Plant peptide hormone signalling. Essays Biochem 58:115–131CrossRefGoogle Scholar
  46. Nambara E, Marion-Poll A (2005) Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol 56:165–185CrossRefGoogle Scholar
  47. Nehela Y, Hijaz F, Elzaawely AA et al (2016) Phytohormone profiling of the sweet orange (Citrus sinensis (L.) Osbeck) leaves and roots using GC-MS-based method. J Plant Physiol 199:12–17CrossRefGoogle Scholar
  48. Nehela Y, Hijaz F, Elzaawely AA et al (2018) Citrus phytohormonal response to Candidatus Liberibacter asiaticus and its vector Diaphorina citri. Physiol Mol Plant Pathol 102:24–35CrossRefGoogle Scholar
  49. Nitsch L, Kohlen W, Oplaat C et al (2012) ABA-deficiency results in reduced plant and fruit size in tomato. J Plant Physiol 169:878–883CrossRefGoogle Scholar
  50. Norris SR, Barrette TR, DellaPenna D (1995) Genetic dissection of carotenoid synthesis in arabidopsis defines plastoquinone as an essential component of phytoene desaturation. Plant Cell 7:2139–2149Google Scholar
  51. Norris SR, Shen X, DellaPenna D (1998) Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxyphenylpyruvate dioxygenase. Plant Physiol 117:1317–1323CrossRefGoogle Scholar
  52. Piotrowska-Niczyporuk A, Bajguz A (2014) The effect of natural and synthetic auxins on the growth, metabolite content and antioxidant response of green alga Chlorella vulgaris (Trebouxiophyceae). Plant Growth Regul 73:57–66CrossRefGoogle Scholar
  53. Porter NG, Van Steveninck RFM (1966) An abscission-promoting factor in Lupinus luteus (L.). Life Sci 5:2301–2308CrossRefGoogle Scholar
  54. Qin G, Gu H, Ma L et al (2007) Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis. Cell Res 17:471–482CrossRefGoogle Scholar
  55. Raschke K, Zeevaart JA (1976) Abscisic acid content, transpiration, and stomatal conductance as related to leaf age in plants of Xanthium strumarium L. Plant Physiol 58:169–174CrossRefGoogle Scholar
  56. Rashad FM, Fathy HM, El-Zayat AS, Elghonaimy AM (2015) Isolation and characterization of multifunctional Streptomyces species with antimicrobial, nematicidal and phytohormone activities from marine environments in Egypt. Microbiol Res 175:34–47CrossRefGoogle Scholar
  57. Rock CD, Sun X (2005) Crosstalk between ABA and auxin signaling pathways in roots of Arabidopsis thaliana (L.) Heynh. Planta 222:98–106CrossRefGoogle Scholar
  58. Rock CD, Zeevaart JA (1991) The aba mutant of Arabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis. Proc Natl Acad Sci USA 88:7496–7499CrossRefGoogle Scholar
  59. Romero I, Tikunov Y, Bovy A (2011) Virus-induced gene silencing in detached tomatoes and biochemical effects of phytoene desaturase gene silencing. J Plant Physiol 168:1129–1135CrossRefGoogle Scholar
  60. Romero P, Gandía M, Alférez F (2013) Interplay between ABA and phospholipases A2 and D in the response of citrus fruit to postharvest dehydration. Plant Physiol Biochem 70:287–294CrossRefGoogle Scholar
  61. Ross GS, Elder PA, McWha JA et al (1987) The development of an indirect enzyme linked immunoassay for abscisic Acid. Plant Physiol 85:46–50CrossRefGoogle Scholar
  62. Rymen B, Sugimoto K (2012) Tuning growth to the environmental demands. Curr Opin Plant Biol 15:683–690CrossRefGoogle Scholar
  63. Salomon MV, Bottini R, de Souza Filho GA et al (2014) Bacteria isolated from roots and rhizosphere of Vitis vinifera retard water losses, induce abscisic acid accumulation and synthesis of defense-related terpenes in in vitro cultured grapevine. Physiol Plant 151:359–374CrossRefGoogle Scholar
  64. Samet JS, Sinclair TR (1980) Leaf senescence and abscisic acid in leaves of field-grown soybean. Plant Physiol 66:1164–1168CrossRefGoogle Scholar
  65. Santner A, Calderon-Villalobos LIA, Estelle M (2009) Plant hormones are versatile chemical regulators of plant growth. Nat Chem Biol 5:301–307CrossRefGoogle Scholar
  66. Santos CAF, Senalik D, Simon PW (2005) Path analysis suggests phytoene accumulation is the key step limiting the carotenoid pathway in white carrot roots. Genet Mol Biol 28:287–293CrossRefGoogle Scholar
  67. Sharp RE, LeNoble ME, Else MA et al (2000) Endogenous ABA maintains shoot growth in tomato independently of effects on plant water balance: evidence for an interaction with ethylene. J Exp Bot 51:1575–1584CrossRefGoogle Scholar
  68. Shu K, Zhou W, Chen F et al (2018) Abscisic acid and gibberellins antagonistically mediate plant development and abiotic stress responses. Front Plant Sci 9:416CrossRefGoogle Scholar
  69. Siewers V, Kokkelink L, Smedsgaard J, Tudzynski P (2006) Identification of an abscisic acid gene cluster in the grey mold Botrytis cinerea. Appl Environ Microbiol 72:4619–4626CrossRefGoogle Scholar
  70. Spence CA, Lakshmanan V, Donofrio N, Bais HP (2015) Crucial roles of abscisic acid biogenesis in virulence of rice blast fungus Magnaporthe oryzae. Front Plant Sci 6:1082CrossRefGoogle Scholar
  71. Srinivasan R, Babu S, Gothandam KM (2017) Accumulation of phytoene, a colorless carotenoid by inhibition of phytoene desaturase (PDS) gene in Dunaliella salina V-101. Bioresour Technol 242:311–318CrossRefGoogle Scholar
  72. Tang J, Han Z, Chai J (2016) Q&A: what are brassinosteroids and how do they act in plants? BMC Biol 14:113CrossRefGoogle Scholar
  73. Wang D, Gao Z, Du P et al (2016) Expression of ABA metabolism-related genes suggests similarities and differences between seed dormancy and bud dormancy of peach (Prunus persica). Front Plant Sci 6:1248Google Scholar
  74. Ward JH (1963) Hierarchical grouping to optimize an objective function. J Am Stat Assoc 58:236–244CrossRefGoogle Scholar
  75. Wei X, Chen C, Yu Q et al (2014a) Comparison of carotenoid accumulation and biosynthetic gene expression between Valencia and Rohde Red Valencia sweet oranges. Plant Sci 227:28–36CrossRefGoogle Scholar
  76. Wei X, Chen C, Yu Q et al (2014b) Novel expression patterns of carotenoid pathway-related genes in citrus leaves and maturing fruits. Tree Genet Genomes 10:439–448CrossRefGoogle Scholar
  77. Yao C, Finlayson SA (2015) Abscisic acid is a general negative regulator of Arabidopsis axillary bud growth. Plant Physiol 169:611–626CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Plant Pathology, Citrus Research and Education Center, IFASUniversity of FloridaLake AlfredUSA

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