Os04g0612000

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OsKS2 gene is a chromosome 4-located ent-kaurene synthase (KS), encoding the enzyme that catalyses an early step of the GA biosynthesis pathway. [1]

Annotated Information

Function

The GA biosynthesis pathway and RT-PCR analysis of the 22 candidate GA metabolic pathway genes in Dongjin and dwarf2. (from reference [1]).

Gibberellins play a fundamental role in regulating several developmental processes, e.g. seed germination [2] and flowering [3]. During germination, GA promotes embryo growth, concurrent with a reduction in the physical restraint imposed by the endosperm and testa, allowing radical protrusion [2]. During flowering, GA is fundamental to development of stamens and petals [4]. Brassinosteroids (BRs), a class of steroidal plant hormone, are widely distributed in both lower and higher plants [5]. BRs are integrated in a complex signalling network, and numerous BR effects appear to be mediated via modulation of levels and sensitivities of other phytohormones. In previous reports, BR activity was demonstrated in almost all auxin and in selected GA bioassays [6][7](. he inductions of α-amylase and shoot elongation by GA, both of which are GA-mediated physiological processes, are classical model systems for studying how GAs act [8][9]. For detection of α-amylase activity, halved seeds are used, and only seeds secreting α-amylase form transparent halos around the seed, resulting from the digestion of starch by α-amylase [10]. The dwarf2 mutant has a defective OsKS2 gene, which is an early step in the GA biosynthesis pathway in rice. The mutant phenotype was restored similar the wild type after exogenous application of bioactive GA3 2 weeks later. Previously, Margis-Pinheiro et al. [11] suggested that mutant seedlings respond similar to the wild type when treated with exogenous GA3. The mutant exhibits multiple abnormal phenotypes: dwarfism, short, wide and dark green leaf blades, reduced tiller number, short roots and early reduction in shoot growth at the seedling stage. In the wild type the internal structures, such as motor cells, large and small vascular bundles and phloem, presented a regular pattern and normal development of leaf blades and elongated stems, whereas in the dwarf mutant there was dramatic modification, including large lacunae, loss of small vascular bundles and submerged lacunae instead of normal, small and large vascular bundles and air spaces in the elongated stem. In rice, GA signalling pathway genes have been reported to regulate fertilisation. Chhun et al. [12] blocked the expression of OsCPS upstream of the GA pathway, which directly affected rice fertilisation and pollen development. However, when the OsKAO gene downstream of the GA pathway was blocked, the resulting mutant exhibited a severe dwarf phenotype and barely developed floral organs. One of the best-known functions of GAs in plants is promotion of stem elongation [2]. Although the number of naturally occurring GAs is large, the number of biologically active GAs is quite small (e.g. GA1, GA3, GA4, GA7 and a few others). Other GAs are either intermediates in the biosynthetic pathway or exist in inactivate forms. Most dicots and some monocots respond by growing faster when treated with GAs, but several species in the Pinaceae show few or no elongation responses to GA3 [13]. However, they do respond well to a mixture of GA4 and GA7 [14]. Short bush beans become climbing pole beans, and dwarf genetic mutants of rice, maize and peas phenotypically exhibit the tall characteristics of normal varieties when treated with GA3 [15]. Terpenes, or terpenoids, are a large class of plant secondary products with a major role in defence against plant-feeding insects and herbivores [16]. However, not all terpenoids act as secondary products, e.g. those involved in photosynthesis, stability of cell membranes, signalling and in biosynthesis of several plant hormones. OsKSL1, OsKSL3 and OsKS2 interact with each other and are involved in the catalytic reactions of GA biosynthesis. The differences between vascular bundles of leaf blade tips in osks2 and Dongjin might cause the curly phenotype of leaf blades, because motor cells play an important role in leaf rolling [17]. The difference of plant growth between Dongjin and osks2 was caused by differences in the shape and cell size of xylem, phloem, motor and mesophyll cells. The abnormal osks2 cell shapes and disorganised cell arrangements may be caused by a defect in their synchronous division and elongation [18]. The OsKS2 mRNA transcripts of Dongjin encoded a putative KS after treatment with UV, salinity, drought or wounding. In particular, expression was higher in salinity- and drought-treated samples than in UV-treated and wounded samples. Moreover, the OsKS2 gene was predominantly expressed under environmental stress.

Mutation

Morphological variations in Dongjin and the dwarf mutant. (from reference [1]).

Among the dwarf mutants whose leaves and stems are externally treated with 50 μm GA3 for 2 weeks, three dwarf mutant lines (dwarf1, dwarf2 and dwarf3) have a restored phenotype similar to the wild type, Dongjin. These dwarf mutants went through a normal vegetative growth stage and exhibited relatively smaller organs compared to Dongjin at various growth stages. The dwarf mutant showed reduced shoot growth, less crown roots and reduced growth of branch roots compared to Dongjin. Additionally, the dwarf mutants showed abnormal leaf blade morphology, with dark-green leaves, shorter and wider edges and leaf tips more rounded than in the wild-type Dongjin. The dwarf mutant has almost sterile seeds, and rapid stem elongation occurred about 2 weeks later. To confirm identification of Ac/Ds inserted dwarfs, Ac/Ds genotype PCR is performed using Ac/Ds gene-specific primers, while the DNA blot analysis of Ds elements is performed using a gene-trap Ds in three dwarf lines. The gene-trap Ds construct contained the 1.2 kb GUS coding region as a reporter [19], and DNA from this region is used as a probe [20].

Expression

mRNA expression of OsKS2. (from reference [1]).

The CPS-like and KS-like genes are considered likely to be involved in diterpene phytoalexin biosynthesis in response to pathogen infection and UV irradiation [21]. Since OsKSL genes are usually induced by environmental stresses, The mRNA expression pattern of OsKS2 in response to various environmental stresses. Northern blot analysis showed that mRNA expression of OsKS2 is induced by salinity (twofold) and drought (four-fold). However, there is no significant increase in mRNA levels after UV treatment and mechanical wounding. OsKS2 transcripts are strongly expressed in salinity and drought compared to the untreated control. An OsKS2 transcript of about 1.8 kb is detected in Dongjin leaves. An 800-bp fragment of the OsKS2 transcript is used as a probe. A total of 15 μg RNA is isolated from different plant parts (i.e. leaf, stem or root) during growth for 30 and 60 days in the wild type. During investigation of the time-course pattern of OsKS2 gene expression, a 2.7-kb transcript of OsKS2 is more strongly detected in leaves, stems and roots of 30-day-old Dongjin than in 60-day-old Dongjin. Generally, the expression of OsKS2 is higher in 30-day-old than 60-day-old plants when probed with 800 bp OsKS2 cDNA at high stringency. The mRNA expression pattern of OsKS2 in various organs of 30-day-old wild-type Dongjin and found it is expressed in all organs – leaf blades, stem, root, callus, flowers and panicles at flowering stage. The OsKS2 mRNA indicated significantly higher expression in the stem, followed by flowers and panicles, whereas expression in leaf, root and callus is similar. These variations in OsKS2 expression at different stages clearly indicate its involvement in regulating the growth period of the plants, which warrants further investigation.

Evolution

Terpene cyclase domain of OsKSL1, OsKS2 and OsKSL3 proteins. (from reference [1]).

The OsKS family genes are not broadly conserved by function. Especially OsKSL1, OsKSL3, OsKSL7 and OsKS2 proteins are more closely related than other proteins. All of the OsKSL family proteins shared significant homology (29–47%) to the OsKS2 protein. The OsKS2 protein has high homology with OsKSL1, sharing 47% identity at the amino acid level. However, relatively lower homology is found between OsKSL3 and OsKS2, with only 29% amino acid sequence identity.

Labs working on this gene

1. Department of Molecular Biotechnology, Konkuk University, Seoul, Korea 2. Subtropical Horticulture Research Institute, Faculty of Biotechnology, Jeju National University, Jeju, Korea 3. Smart Bio Lab Co., Ltd., Suwon, Korea 4. National Academy of Agricultural Science, RDA, Suwon, Korea

References

  1. ↑ 1.0 1.1 1.2 1.3 1.4 i, S. H., Gururani, M. A., Lee, J. W., Ahn, B.-O., Chun, S.-C. (2014), Isolation and characterisation of a dwarf rice mutant exhibiting defective gibberellins biosynthesis. Plant Biology, 16: 428–439. doi: 10.1111/plb.12069
  2. ↑ 2.0 2.1 2.2 Debeaujon I., Koornneef M. (2000) Gibberellin requirement for Arabidopsis seed germination is determined both by testa characterization and embryonic abscisic acid. Plant Physiology, 122, 415–424.
  3. ↑ King R.W., Evans L.T. (2003) Gibberellins and flowering of grasses and cereals: prizing open the lid of the “Florigen” black box. Annual Review of Plant Biology, 54, 307–328.
  4. ↑ Olszewski N., Sun T.P., Gubler F. (2002) Gibberellin signaling: Biosynthesis, catabolism, and response pathways. The Plant Cell, 14, 61–80.
  5. ↑ Grove M.D., Spencer G.F., Rohwedder W.K., Mandava N.B., Worley J.F., Warthen J.D., Steffens G.L., Flippen-Anderson J.L., Cook J.C. (1979) Brassinolide, a plant growth-promoting steroid isolated from Brassica napus pollen. Nature, 281, 216–217.
  6. ↑ Yopp J.H., Colclasure G.C., Mandava N. (1979) Effects of brassin-complex on auxin and gibberellin mediated events in the morphogenesis of the etiolated bean hypocotyl. Physiologia Plantarum, 46, 247–254.
  7. ↑ Mandava N.B., Sasse J.M., Yopp J.H. (1981) Brassinolide, a growth-promoting steroidal lactone. II. Activity in selected gibberellin and cytokinin bioassays. Physiologia Plantarum, 53, 453–461.
  8. ↑ Lanahan M.B., Ho T.D.H., Rogers S.W., Rogers J.C. (1992) A gibberellin response complex in cereal alpha-amylase gene promoters. The Plant Cell, 4, 203–211.
  9. ↑ Matsukura C., Itoh S., Nemoto K., Tanimoto E., Yamaguchi J. (1998) Promotion of leaf sheath growth by gibberellic acid in a dwarf mutant of rice. Planta, 205, 145–152.
  10. ↑ Lanahan M.B., Ho T.D.H. (1988) Slender barley: a constitutive gibberellin-response mutant. Planta, 175, 107–114.
  11. ↑ Margis-Pinheiro M., Zhou X.R., Zhu Q.H., Dennis E.S., Upadhyaya N.M. (2005) Isolation and characterization of a Ds-tagged rice (Oryza sativa L.) GA-responsive dwarf mutant defective in an early step of the gibberellin biosynthesis pathway. Plant Cell Reports, 23, 819–833.
  12. ↑ Chhun T., Aya K., Asano K., Yamamoto E., Morinaka Y., Watanabe M., Kitano H., Ashikari M., Matsuoka M., Ueguchi-Tanaka M. (2007) Gibberellin regulates pollen viability and pollen tube growth in rice. The Plant Cell, 19, 3876–3888.
  13. ↑ Pharisr P., Kuo C.G. (1977) Physiology of gibberellins in conifers. Canadian Journal of Forest Research, 7, 299–325.
  14. ↑ Pharis R.P., Evans L.T., King R.W., Mander L.N. (1989) Gibberellins and Flowering in Higher Plants – Differing structures yield highly specific effects. In: Lord E., Bernier G. (Eds), Plant reproduction: from floral induction to pollination, 1. Symposium of the American Society of Plant Physiology, 29–41.
  15. ↑ Nishijima T., Katsuma N. (1989) A modified micro-drop bioassay using dwarf rice for detection of femtomol quantities of gibberellins. Plant & Cell Physiology, 30, 623–627.
  16. ↑ Yamaguchi S., Sun T., Kawaide H., Kamiya Y. (1998) The GA2 locus of Arabidopsis thaliana encodes ent-kaurene synthase of gibberellin biosynthesis. Plant Physiology, 116, 1271–1278.
  17. ↑ Hoshikawa K. (1989) The growing rice plant—an anatomical monograph. Nobunkyo Press, Tokyo, Japan.
  18. ↑ Komorisono M., Ueguchi-Tanaka M., Aichi I., Hasegawa Y., Ashikari M., Kitano H., Matsuoka M., Sazuka T. (2005) Analysis of rice mutant dwarf and gladius leaf 1. Aberrant katanin-mediated microtubule organization genes independently of gibberellin signaling. Plant Physiology, 138, 1982–1993.
  19. ↑ Chin H.G., Choe M.S., Lee S.H., Park S.H., Koo J.C., Kim N.Y., Lee J.J., Oh B.G., Yi G.H., Kim S.C., Choi H.C., Cho M.J., Han C.D. (1999) Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system. The Plant Journal, 19, 615–623.
  20. ↑ Park S.H., Jun N.S., Kim C.M., Oh T.Y., Huang J., Xuan Y.H., Park S.J., Je B.I., Piao H.L., Park S.H., Cha Y.S., Ahn B.O., Ji H.S., Lee M.C., Suh S.C., Nam M.H., Eun M.Y., Yi G.H., Yun D.W., Han C.D. (2007) Analysis of gene-trap DS rice populations in Korea. Plant Molecular Biology, 65, 373–384.
  21. ↑ Sakamoto T., Miura K., Itoh H., Tatsumi T., Ueguchi-Tanaka M., Ishiyama K., Kobayashi M., Agrawal G.K., Takeda S., Abe K., Miyao A., Hirochika H., Kitano H., Ashihikara M., Matsuoka M. (2004) An overview of gibberellin metabolism enzyme genes and their related mutants in rice. Plant Physiology, 134, 1642–1653.

Structured Information

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