Os06g0662000

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A rice (Oryza sativa L.) vacuolar H+ -ATPase subunit A (OsVHA-A) gene plays an important role in the regulation of stomatal movement and determination of stomatal density[1].

Annotated Information

Function

Figure 1. Schematical model of OsVHA-A-RNAi in the regulation of stomatal aperture.(from reference [1]).
  • Using reverse transcription PCR, a 1863 bp cDNA fragment containing a complete open reading frame (ORF) was obtained and designated as OsVHA-A (accession no. NM_001064815.1)[1].
  • OsVHA-A(Os06g0662000) encoded a protein of 620 amino acids with a predicted molecular weight of 65.62 kDa revealed by Sequence analysis[1].
  • By using inverse genetics approach, Zhang et al. demonstrated that OsVHA-A plays an important role in the regulation of stomatal movement and determination of stomatal density, which is associated with increase or inhibition of growth of rice plants under nonstress or salt/osmotic stress conditions, respectively[1].
  • Knockdown of OsVHA-A in the transgenic plants specifically suppressed the V-ATPase activity instead of V-PPase activity. The elevation of vacuolar pH value in OsVHA-A RNAi transgenic lines might be attributed to the reduction of V-ATPase proton-pumping activity. Knockdown of OsVHA-A triggers the H+ efflux and the hyperpolarization of plasma membrane, which enhances the uptake of K+ , resulting in the expand of stomatal aperture[1].
  • As shown in Figure 1, a possible signaling pathway to decipher the nature of OsVHA-A RNAi-induced increase of stomatal conductance was presented. Repression of OsVHA-A expression may lead to a decrease of V-ATPase activity. The decrease of this enzyme activity promoted PMA3 gene expression and activated the pump of plasma membrane H+-ATPase, resulting in the acceleration of H+ efflux and K+ influx. Moreover, the decrease of V-ATPase activity resulted in downregulation of CAM1, CAM3 and YDA1 that have been demonstrated to be involved in regulation of stomatal opening and density. OsVHA-A may control the stomatal conductance via regulating ionic equilibrium of proton pump and downstream gene expression.

GO assignment(s): GO:0005524, GO:0006754, GO:0015986, GO:0016469

Mutation

vma1△:

  • To further test the biochemical activity of OsVHA-A, the yeast(Saccharomyces cerevisiae) mutant (vma1△) lacking subunit A of VATPase was employed for complementation assay[2].
  • OsVHA-A RNAi transgenic lines[1]:
    • OsV-5
    • OsV-11
    • OsV-18

No significant difference in the V-PPase activity was detected between WT and OsVHA-A RNAi transgenic plants, which suggesting that knockdown of OsVHA-A in the transgenic plants specifically suppressed the V-ATPase activity instead of V-PPase activity.

Vacuoles from the transgenic root cells had a pH fluctuation ranging from 6.7 to 6.9, whereas the pH value in WT root cells was about 6.2, indicating that knockdown of OsVHA-A' inhibited the transport of H+ from cytosol into vacuole.

The V-ATPase proton-pumping activity in three OsVHA-A RNAi transgenic lines were markedly lower than that in WT, indicating that the elevation of vacuolar pH value in OsVHA-A RNAi transgenic lines might be attributed to the reduction of V-ATPase proton-pumping activity.

Expression

  • Differential expression levels of OsVHA-A gene were detected in root, stem, leaf, flower and internode from the mature rice plant. The result indicated OsVHA-A was expressed constitutively, with greater expression abundance in leaf and flower. Heterologous expression of OsVHA-A appears to partially complement the defective V-ATPase activity in the yeast vma1△ mutant[1][2].
  • Knockdown of OsVHA-A Promotes the H+ Efflux by Increasing Plasma Membrane H+-ATPase Activity. Knockdown of OsVHA-A leads to remarkably increased stomatal aperture and density[1].
  • OsVHA-A RNAi repression lines shows a significantly increased sensitivity to salt stress. The enhanced sensitivity of OsVHA-A RNAi lines might be due to an accumulation of Na+ toxicity as well as a disruption of osmotic adjustment.OsVHA-A RNAi repression lines have significantly increased sensitivity to osmotic stress. The enhanced drought sensitivity in RNAi lines is due to a low solute potential. OsVHA-A-RNAi suppressed the closure of stomata in response to stress conditions[1].

Subcellular localization

According to the subcellular localization of OsVHA-A, green fluorescence signal derived from GFP-OsVHAA fusion protein was exclusively detected in tonoplast, while green fluorescence signal from GFP alone was visualized throughout the cytoplasm and nucleus[1].

Evolution

Figure 2. Phylogenetic analysis of OsVHA-A homologues.(from reference [1]).

Amino acid alignment showed that OsVHA-A shared 95.40%, 94.77%, 91.92% and 89.70% identity with the orthologs from Sorghum bicolor (accession no. XM_002451594.1), Zea mays (accession no.AY104754.1), Triticum aestivum (accession no. AK332978.1), Arabidopsis thaliana (accession no. NM_001036222.2), respectively, and the homologous proteins derived from Oryza sativa, Sorghum bicolor, Zea mays were closely clustered, whereas those from other species formed several evolutionary branches(Figure 2).

Knowledge Extension

Stomatal pores is surrounded by a pair of guard cells, which play a crucial role in controlling gaseous exchange and water release by transpiration[3]. The development of stomata and the regulation of stomatal apertures are critical for plant survival and productivity. Stomatal aperture is regulated by the reversible swelling and shrinking of guard cells, which sense environmental signals and endogenous hormonal stimuli, such as light, atmospheric CO2 levels, humidity, temperature, pathogens and hormones[3][4]. In response to these stimuli, transport of ions and water through channel proteins across the plasma and vacuolar membranes changes the turgor and volume of guard cell, thereby regulating stomatal aperture[5].

Subunit A, the critical component of V-ATPase protein complex, contains an ATP-binding region and may represent a catalytic reaction center[6]. The transcript level of subunit A of V-ATPase has been shown previously to be induced by salt and osmotic stresses in Arabidopsis and barley[7][8].

Labs working on this gene

  • School of Life Science, Chongqing University, Chongqing, China
  • School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, China,
  • Department of Plant, Soil, and Entomological Sciences, University of Idaho Moscow, Idaho, United States of America
  • Ministry of Education Key Laboratory for Bioresource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu, China

References

  1. 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 Zhang H, Niu X, Liu J, et al. RNAi-directed downregulation of vacuolar H+-ATPase subunit a results in enhanced stomatal aperture and density in rice[J]. PloS one, 2013, 8(7): e69046.
  2. 2.0 2.1 Kim W, Wan C Y, Wilkins T A. Functional complementation of yeast vma1Δ cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H+‐ATPase activity[J]. The Plant Journal, 1999, 17(5): 501-510.
  3. 3.0 3.1 Assmann S M. Signal transduction in guard cells[J]. Annual review of cell biology, 1993, 9(1): 345-375.
  4. Hetherington A M, Woodward F I. The role of stomata in sensing and driving environmental change[J]. Nature, 2003, 424(6951): 901-908.
  5. Kim T H, Maik B Ć. Guard cell signal transduction network: advances in understanding abscisic acid, CO2, and Ca2+ signaling[J]. Annual review of plant biology, 2010, 61: 561.
  6. Maher M J, Akimoto S, Iwata M, et al. Crystal structure of A3B3 complex of V‐ATPase from Thermus thermophilus[J]. The EMBO journal, 2009, 28(23): 3771-3779.
  7. Magnotta S M, Gogarten J P. Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana[J]. BMC plant biology, 2002, 2(1): 3.
  8. Fukuda A, Chiba K, Maeda M, et al. Effect of salt and osmotic stresses on the expression of genes for the vacuolar H+‐pyrophosphatase, H+‐ATPase subunit A, and Na+/H+ antiporter from barley*[J]. Journal of Experimental Botany, 2004, 55(397): 585-594.

Structured Information