Skip to main content
Log in

Downregulation of OsPK1, a cytosolic pyruvate kinase, by T-DNA insertion causes dwarfism and panicle enclosure in rice

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

Pyruvate kinase (PK) catalyzes the final step of glycolysis. There are few reports on the role of PK in rice. Here, we identified a novel rice dwarf mutant, designated as ospk1, showing dwarfism, panicle enclosure, reduced seed set, and outgrowth of axillary buds from culm nodes. Sequence analyses of 5′-RACE indicated that a single T-DNA was inserted in the transcriptional regulatory region of OsPK1 in ospk1. Quantitative RT-PCR result showed that OsPK1 expression was decreased by approximately 90% in ospk1 compared with that in WT. Enzyme assay and transient expression in protoplasts indicated that OsPK1 encodes a cytosolic PK (PKc). Complementation with OsPK1 demonstrated that OsPK1 is responsible for the phenotype of ospk1. Quantitative RT-PCR and GUS staining analyses exhibited that OsPK1 was expressed mainly in leaf mesophyll cells, phloem companion cells in stems, and cortical parenchyma cells in roots. The transcriptions of four other putative enzymes involved in the glycolysis/gluconeogenesis pathway were altered in ospk1. The amount of pyruvate is decreased in ospk1. We propose that OsPK1 plays an important role through affecting the glycolytic pathway. The contents of glucose and fructose were markedly accumulated in flag leaf blade and panicle of ospk1. The sucrose level in panicle of ospk1 was decreased by approximately 84%. These findings indicated that both monosaccharide metabolism and sugar transport are altered due to the decreased expression of OsPK1. Together, these results provide new insights into the role of PKc in plant morphological development, especially plant height.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

A 340 :

Absorbance at 340 nm

ALT:

Alanine transaminase

GC-MS:

Gas chromatography-mass spectrometry

Int:

Internode

ME:

Malic enzyme

OAA:

Oxaloacetate

PDH:

Pyruvate dehydrogenase

PEP:

Phosphoenolpyruvate

PEPC:

PEP carboxylase

PEPCK:

PEP carboxykinase

PK:

Pyruvate kinase

PKc and PKp :

Cytosolic and plastid PK isozymes, respectively

RACE:

Rapid amplification of cDNA ends

RT-PCR:

Reverse transcription polymerase chain reaction

TAIL-PCR:

Thermal asymmetric interlaced-PCR

TCA:

Tricarboxylic acid

TIM:

Triosephosphate isomerase

UI:

Uppermost internode

References

  • Andre C, Froehlich JE, Moll MR, Benning C (2007) A heteromeric plastidic pyruvate kinase complex involved in seed oil biosynthesis in Arabidopsis. Plant Cell 19:2006–2022

    Article  PubMed  CAS  Google Scholar 

  • Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815

    Article  Google Scholar 

  • Baud S, Wuilleme S, Dubreucq B, de Almeida A, Vuagnat C, Lepiniec L, Miquel M, Rochat C (2007) Function of plastidial pyruvate kinases in seeds of Arabidopsis thaliana. Plant J 52:405–419

    Article  PubMed  CAS  Google Scholar 

  • Borisjuk L, Rolletschek H, Radchuk R, Weschke W, Wobus U, Weber H (2004) Seed development and differentiation: a role for metabolic regulation. Plant Biol 6:375–386

    Article  PubMed  CAS  Google Scholar 

  • Cole KP, Blakeley SD, Dennis DT (1992) Structure of the gene encoding potato cytosolic pyruvate-kinase. Gene 122:255–261

    Article  PubMed  CAS  Google Scholar 

  • Dai MQ, Zhao Y, Ma Q, Hu YF, Hedden P, Zhang QF, Zhou DX (2007) The rice YABBY1 gene is involved in the feedback regulation of gibberellin metabolism. Plant Physiol 144:121–133

    Google Scholar 

  • Drincovich MF, Casati P, Andreo CS (2001) NADP-malic enzyme from plants: a ubiquitous enzyme involved in different metabolic pathways. FEBS Lett 490:1–6

    Article  PubMed  CAS  Google Scholar 

  • Ebbighausen H, Chen J, Heldt HW (1985) Oxaloacetate translocator in plant mitochondria. Biochim Biophys Acta 810:184–199

    Article  CAS  Google Scholar 

  • Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunesekaran P, Ceric G, Forslund K, Holm L, Sonnhammer EL, Eddy SR, Bateman A (2010) The Pfam protein families database. Nucl Acids Res 38:D211–D222

    Article  PubMed  CAS  Google Scholar 

  • Grodzinski B, Jiao J, Knowles VL, Plaxton WC (1999) Photosynthesis and carbon partitioning in transgenic tobacco plants deficient in leaf cytosolic pyruvate kinase. Plant Physiol 120:887–895

    Article  PubMed  CAS  Google Scholar 

  • Hausler RE, Fischer KL, Flugge UI (2000) Determination of low-abundant metabolites in plant extracts by NAD(P)H fluorescence with a microtiter plate reader. Anal Biochem 281:1–8

    Article  PubMed  CAS  Google Scholar 

  • Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282

    Article  PubMed  CAS  Google Scholar 

  • Hu ZY, Plaxton WC (1996) Purification and characterization of cytosolic pyruvate kinase from leaves of the castor oil plant. Arch Biochem Biophys 333:298–307

    Article  PubMed  CAS  Google Scholar 

  • Jenner HL, Winning BM, Millar AH, Tomlinson KL, Leaver CJ, Hill SA (2001) NAD malic enzyme and the control of carbohydrate metabolism in potato tubers. Plant Physiol 126:1139–1149

    Article  PubMed  CAS  Google Scholar 

  • Kayne FJ, Price NC (1973) Amino acid effector binding to rabbit muscle pyruvate kinase. Arch Biochem Biophys 159:292–296

    Article  PubMed  CAS  Google Scholar 

  • Knowles VL, McHugh SG, Hu ZY, Dennis DT, Miki BL, Plaxton WC (1998) Altered growth of transgenic tobacco lacking leaf cytosolic pyruvate kinase. Plant Physiol 116:45–51

    Article  PubMed  CAS  Google Scholar 

  • Kromer S, Gardestrom P, Samuelsson G (1996) Regulation of the supply of cytosolic oxaloacetate for mitochondrial metabolism via phosphoenolpyruvate carboxylase in barley leaf protoplasts: I. The effect of covalent modification on PEPC activity, pH response, and kinetic properties. Biochim Biophys Acta 1289:343–350

    Article  PubMed  CAS  Google Scholar 

  • Kubis SE, Pike MJ, Hill LM, Rawsthorne S (2004) The import of phosphoenolpyruvate by plastids from developing embryos of oilseed rape Brassica napus (L.), and its potential as a substrate for fatty acid synthesis. J Exp Bot 55:1455–1462

    Article  PubMed  CAS  Google Scholar 

  • Li XY, Qian Q, Fu ZM, Wang YH, Xiong GS, Zeng DL, Wang XQ, Liu XF, Teng S, Hiroshi F, Yuan M, Luo D, Han B, Li JY (2003) Control of tillering in rice. Nature 422:618–621

    Article  PubMed  CAS  Google Scholar 

  • Li XY, Lin HQ, Zhang WG, Zou Y, Zhang J, Tang XY, Zhou JM (2005) Flagellin induces innate immunity in nonhost interactions that is suppressed by Pseudomonas syringae effectors. Proc Natl Acad Sci USA 102:12990–12995

    Article  PubMed  CAS  Google Scholar 

  • Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8:457–463

    Article  PubMed  CAS  Google Scholar 

  • Malcovati M, Valentini G, Kornberg HL (1973) Two forms of pyruvate kinase in E. coli: their properties and regulation. Acta Vitaminol Enzymol 27:96–111

    PubMed  CAS  Google Scholar 

  • McHugh SG, Knowles VL, Blakeley SD, Sangwan RS, Miki BL, Dennis DT, Plaxton WC (1995) Differential expression of cytosolic and plastid pyruvate kinase isozymes in tobacco. Physiol Plant 95:507–514

    Article  CAS  Google Scholar 

  • Munoz ME, Ponce E (2003) Pyruvate kinase: current status of regulatory and functional properties. Comp Biochem Physiol B Biochem Mol Biol 135:197–218

    Article  PubMed  Google Scholar 

  • Oliver SN, Lunn JE, Urbanczyk-Wochniak E, Lytovchenko A, van Dongen JT, Faix B, Schmalzlin E, Fernie AR, Geigenberger P (2008) Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase. Plant Physiol 148:1640–1654

    Article  PubMed  CAS  Google Scholar 

  • Plaxton WC (1989) Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isoenzymes from castor-oil-plant endosperm and leaf. Eur J Biochem 181:443–451

    Article  PubMed  CAS  Google Scholar 

  • Plaxton WC (1996) The organization and regulation of plant glycolysis. Annu Rev Plant Physiol Plant Mol Biol 47:185–214

    Article  PubMed  CAS  Google Scholar 

  • Plaxton WC, Podesta FE (2006) The functional organization and control of plant respiration. Crit Rev Plant Sci 25:159–198

    Article  CAS  Google Scholar 

  • Plaxton WC, Smith CR, Knowles VL (2002) Molecular and regulatory properties of leucoplast pyruvate kinase from Brassica napus (rapeseed) suspension cells. Arch Biochem Biophys 400:54–62

    Article  PubMed  CAS  Google Scholar 

  • Sha Y, Li S, Pei Z, Luo L, Tian Y, He C (2004) Generation and flanking sequence analysis of a rice T-DNA tagged population. Theor Appl Genet 108:306–314

    Article  PubMed  CAS  Google Scholar 

  • Tan YP, Li K, Hu L, Chen S, Gai Y, Jiang XN (2010) Fast and simple droplet sampling of sap from plant tissues and capillary microextraction of soluble saccharides for picogram-scale quantitative determination with GC–MS. J Agric Food Chem 58:9931–9935

    Article  PubMed  CAS  Google Scholar 

  • Tang GQ, Hardin SC, Dewey R, Huber SC (2003) A novel C-terminal proteolytic processing of cytosolic pyruvate kinase, its phosphorylation and degradation by the proteasome in developing soybean seeds. Plant J 34:77–93

    Article  PubMed  CAS  Google Scholar 

  • Turner WL, Plaxton WC (2000) Purification and characterization of cytosolic pyruvate kinase from banana fruit. Biochem J 352:875–882

    Article  PubMed  CAS  Google Scholar 

  • Turner WL, Knowles VL, Plaxton WC (2005) Cytosolic pyruvate kinase: subunit composition, activity, and amount in developing castor and soybean seeds, and biochemical characterization of the purified castor seed enzyme. Planta 222:1051–1062

    Article  PubMed  CAS  Google Scholar 

  • Van Bel AJE (2003) The phloem, a miracle of ingenuity. Plant Cell Environ 26:125–149

    Article  Google Scholar 

  • Wang SM, Lue WL, Yu TS, Long JH, Wang CN, Eimert K, Chen J (1998) Characterization of ADG1, an Arabidopsis locus encoding for ADPG pyrophosphorylase small subunit, demonstrates that the presence of the small subunit is required for large subunit stability. Plant J 13:63–70

    Article  PubMed  CAS  Google Scholar 

  • Yamada K, Noguchi T (1999) Nutrient and hormonal regulation of pyruvate kinase gene expression. Biochem J 337:1–11

    Article  PubMed  CAS  Google Scholar 

  • Yamamuro C, Ihara Y, Wu X, Noguchi T, Fujioka S, Takatsuto S, Ashikari M, Kitano H, Matsuoka M (2000) Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint. Plant Cell 12:1591–1605

    Article  PubMed  CAS  Google Scholar 

  • Yin CX, Gan LJ, Ng D, Zhou X, Xia K (2007) Decreased panicle-derived indole-3-acetic acid reduces gibberellin A1 level in the uppermost internode, causing panicle enclosure in male sterile rice Zhenshan 97A. J Exp Bot 58:2441–2449

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Xiangning Jiang and Xiaoqiao Cheng of Beijing Forestry University for technical assistance for sugar measurement. This work was supported by grants from the National Natural Science Foundation of China (Grant No. 30571003) and the Natural Science Foundation of Hainan Province (Grant No. 30204).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Chaozu He.

Additional information

Yan Zhang and Wenkai Xiao contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

425_2011_1471_MOESM1_ESM.tif

Supplementary Fig. S1 Characteristics of ospk1. a Non-synchronous development of WT and ospk1 seeds after 6 days of accelerated germination. b Roots of 10-day-old seedlings of WT and ospk1 grown in 1/2 MS medium. c Seedlings (9 days old) of WT and ospk1. d Length of individual internodes of WT and ospk1. 1–5, internodes numbered from top to bottom (TIFF 6290 kb)

425_2011_1471_MOESM2_ESM.tif

Supplementary Fig. S2 Pyruvate kinase phylogeny. Os, Oryza sativa; Zm, Zea mays; Sb, Sorghum bicolor; At, Arabidopsis thaliana. Amino acid sequences of putative pyruvate kinases were used for phylogenetic analysis. Bootstrap frequency values based on 1000 replicates are indicated. Scale represents 10% difference (TIFF 4803 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, Y., Xiao, W., Luo, L. et al. Downregulation of OsPK1, a cytosolic pyruvate kinase, by T-DNA insertion causes dwarfism and panicle enclosure in rice. Planta 235, 25–38 (2012). https://doi.org/10.1007/s00425-011-1471-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-011-1471-3

Keywords

Navigation