Research article
OsGERLP: A novel aluminum tolerance rice gene isolated from a local cultivar in Indonesia

https://doi.org/10.1016/j.plaphy.2021.02.019Get rights and content

Highlights

  • OsGERLP is highly expressed in the rice root tips under aluminum (Al) stress.

  • Silencing OsGERLP in rice decreases the expression of Al tolerance related genes and impairs root growth under Al stress.

  • Overexpression of OsGERLP in tobacco elevates the expression of MATE gene, citrate secretion, and tolerance to Al stress.

  • Thus, the OsGERLP could be a novel Al tolerance gene in rice.

Abstract

There is a decrease in the land available for rice cultivation due to the rapid conversion to urban uses. Subsequently, acid soil could be an alternative land cultivating rice, but will require the use of aluminum (Al)-tolerant rice varieties. This Al tolerance trait is genetically controlled, and there is a need to discover more genes needed to develop Al-tolerant rice. Therefore, the objective of this study was to clone and characterize a novel Al tolerance gene isolated from a local cultivar of Indonesian rice. The gene cloning was conducted based on the rye/rice microsynteny relationship. In addition, the root growth and gene expression analyses were performed to verify the role of the gene on Al tolerance in gene-silenced rice and in overexpressed transgenic tobacco. The results showed an Al tolerance candidate gene, OsGERLP, was successfully cloned from rice cv. Hawara Bunar, with its gene encoding a protein similar to a bacterial ribosomal L32 protein. Additionally, the analysis showed that low gene expression caused the gene-silenced rice to be sensitive to Al, while high expression induced the Al tolerance in transgenic tobacco. Furthermore, it was discovered that the gene expression level in both plants was in line with the lower expression of the OsFRDL4 gene in the silenced rice and the high expression of the MATE gene in transgenic tobacco also with the higher citrate secretion from transgenic tobacco roots. In conclusion, the OsGERLP gene could act as a regulator for other Al tolerance genes, with the potential to develop Al-tolerant rice varieties.

Introduction

Rice (Oryza sativa L.) is an essential staple food in Indonesia and one of the largest food crops in the world. Its intensive cultivation is a common agricultural practice that aims at high yield. However, the decreasing paddy fields due to rapid conversion into settlement and industrial uses significantly limit rice production. An alternative solution to this problem is the extension of rice cultivation area to suboptimal lands, such as acid soil, which is abundant in Indonesia. However, high aluminum (Al) solubility in soil with a pH of less than 5 negatively affects rice cultivation in acid soil (Delhaize and Ryan, 1995; Delhaize et al., 2001). Al ions in the rhizosphere damage the root system (Ma and Hiradate, 2000), making plants more vulnerable to drought and mineral nutrition deficiency. These conditions inhibit growth and decrease production (Matsumoto, 2000).

The use of Al-tolerant varieties adapted to acid soil would significantly solve the Al toxicity problem worldwide. In Indonesia, several local cultivars are tolerant to Al, such as Grogol, Krowal, and Hawara Bunar, though they are not high yielding (Miftahudin et al., 2007; Miftahudin and Chikmawati, 2008). Hawara Bunar is considered the most Al-tolerant rice cultivar. Hence, it can be used to develop high yielding rice varieties tolerant to Al and acid soil.

Several genes control the Al tolerance trait in rice (Nguyen et al., 2003), and some of them have been identified and isolated (Yamaji et al., 2009; Tsutsui et al., 2011; Yokosho et al., 2011; Li et al., 2014). Miftahudin et al. (2005) reported microsynteny between an Al tolerance locus on rye (Secale cereale L.) chromosome arm 4RL and a BAC clone OSJNBa0091P11 from rice chromosome 3. Two markers, BCD1230 and B6, border both regions. Additionally, we identified a potential Al-tolerance candidate gene between markers in the rice BAC clone.

Citrate secretion contributes to the Al tolerance mechanisms in rice (Ma et al., 2005). The OsFRDL4 gene, which is upregulated by Al, controls the citrate secretion from the rice roots (Yokosho et al., 2011). According to Ma et al. (2014), the transcription factor, ART1, controls the OsFRDL4 gene expression. However, further analysis showed that Al did not regulate the ART1 gene. This indicates that an unknown upstream gene may regulate the ART1 gene. To understand Al tolerance mechanisms in rice, it is necessary to clone the new Al tolerance candidate gene from rice chromosome and analyze its role in controlling the Al-tolerance trait and regulating Al-related gene expression.

This paper reports on the cloning and characterization of an Al tolerance candidate gene, OsGERLP (GenBank: MH388027.1). The gene is isolated from rice cv. Hawara Bunar, a local Indonesian rice cultivar, using the microsynteny relationship between rye and rice. The gene's responses of the OsGERLP-silenced transgenic rice to Al stress are evaluated. Furthermore, the role in enhancing Al tolerance in transgenic tobacco (Nicotiana tabacum L.) is also evaluated.

Section snippets

Plant materials

This experiment used Rice cv. Hawara Bunar, IR64, and the F9 Recombinant Inbred Line Population derived between IR64 and Hawara Bunar, as well as T3 transgenic tobacco and its wild-type seeds. The T3 transgenic tobacco seeds with a homozygous OsGERLP gene were obtained from T2 plants. T1 transgenic rice seeds containing 3-UTR of the OsGERLP gene were used to generate OsGERLP-silenced T2 transgenic plants (Wahyuningtyas et al., 2016). The OsGERLP-silenced T2 homozygous plants were then used for

Rice root length characters under aluminum stress

The Al-tolerant rice cv. Hawara Bunar shows a higher TRL than the Al-sensitive rice cv. IR64 (Fig. 1). In previous studies, the TRL and PRL are Al tolerance characters which have be used to distinguish between several Al-tolerant and sensitive local cultivars of Indonesian rice (Siska et al., 2017; Fendiyanto et al., 2019a). To reconfirm the Al tolerance level of rice cv. Hawara Bunar and IR64, they were evaluated using PRL and TRL characters under 555 μM Al stress. Al-treated rice cv. Hawara

Discussion

Rice shows different tolerance levels to Al toxicity among cultivars. Data showed that rice cv. Hawara Bunar is more tolerant to Al than cv. IR64, supported by root length characters, Al accumulation, and citrate secretion data (Fig. 1). Additionally, based on the primary root length (PRL) and total root length (TRL) parameters, rice cv. Hawara Bunar shows only a slight decrease in both root length characteristics between Al-treated and control conditions. Conversely, rice cv. IR64 showed a

Author contributions

M.M. is the PI of the research project, designed and conducted the experiment, drafted, edited, and finalized the manuscript. T.C.H., S.S designed, conducted the experiment, and edited the manuscript. H.T.N. and J.P.G. collaborators, partially designed the experiment, provided facilities and expression vector, and edited the manuscript. D.I.R., A.H. identified, isolated, partially characterized the OsGERLP in rice, and transformed the tobacco. M.H.F. and R.D.S. conducted QTL mapping and

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

The research was funded by the International Collaboration and Scientific Publication Research Grant, F.Y. 2014–2016, and World Class Research (WCR) Grant, F.Y. 2019 awarded to Dr. Miftahudin from the Indonesian Ministry of Research and Technology, the Republic of Indonesia. We thank Dr. Utut Widyastuti for her suggestions and technical assistance in the molecular biology laboratory work.

References (70)

  • J. Che et al.

    Functional characterization of an aluminum (Al)-inducible transcription factor, ART2, revealed a different pathway for Al tolerance in rice

    New Phytol.

    (2018)
  • R.T. Clark et al.

    High-throughput two-dimensional root system phenotyping platform facilitates genetic analysis of root growth and development

    Plant Cell Environ.

    (2013)
  • M.D. Curtis et al.

    A gateway cloning vector set for high-throughput functional analysis of genes in planta

    Plant Physiol.

    (2003)
  • E. Delhaize et al.

    Expression of Pseudomonas aeruginosa citrate synthase gene in tobacco is not associated with either enhanced citrate accumulation of efflux

    Plant Physiol.

    (2001)
  • E. Delhaize et al.

    Aluminum toxicity and tolerance in plant

    Plant Physiol.

    (1995)
  • E. Delhaize et al.

    Engineering high level aluminum tolerance in barley with the ALMT1 gene

    Proc. Natl. Acad. Sci. U.S.A.

    (2004)
  • E. Delhaize et al.

    Aluminum tolerance in wheat (Triticum aestivum L.): II. Aluminum stimulated excretion of malic acid from root apices

    Plant Physiol.

    (1993)
  • E. Delhaize et al.

    Transgenic barley (Hordeum vulgare L.) expressing the wheat aluminum resistance gene (TaALMT1 ) shows enhanced phosphorus nutrition and grain production when grown on an acid soil

    Plant Biotechnol. J.

    (2009)
  • O. Emanuelsson et al.

    Locating proteins in the cell using TargetP, SignalP, and related tools

    Nat. Protoc.

    (2007)
  • B. Ezaki et al.

    Expression of aluminum induced genes in transgenic Arabidopsis plants can ameliorate aluminum stress and/or oxidative stress

    Plant Physiol.

    (2000)
  • D. Eticha et al.

    Localization of aluminium in the maize root apex: can morin detect cell wall-bound aluminium?

    J. Exp. Bot.

    (2005)
  • M.A. Farajzadeh et al.

    Citric acid determination by dual wavelength spectrophotometry

    J. Chil. Chem. Soc.

    (2002)
  • M.H. Fendiyanto et al.

    Correlation among Snpb11 markers, root growth, and physiological characters of upland rice under aluminum stress

    Biodiversitas

    (2019)
  • M.H. Fendiyanto et al.

    QTL for aluminum tolerance on rice chromosome-3 based on root length characters

    SABRAO J. Breed. Genet.

    (2019)
  • C. Huang et al.

    A bacterial-type ABC transporter is involved in aluminum tolerance in rice

    Plant Cell

    (2009)
  • S. Iuchi et al.

    Zinc finger protein STOP1 is critical for proton tolerance in Arabidopsis and coregulates a key gene in aluminum tolerance

    Proc. Natl. Acad. Sci. U.S.A.

    (2007)
  • D.A. Johansen

    Plant Microtechnique

    (1940)
  • H.D. Jones et al.

    Transgenic Wheat, Barley, and Oats: Production and Characterization Protocols

    (2009)
  • L. Käll et al.

    Advantages of combined transmembrane topology and signal peptide prediction the Phobius web server

    Nucleic Acids Res.

    (2007)
  • F. Kempken et al.

    Genetic modification of plants: agriculture, horticulture and forestry

  • L.V. Kochian

    Cellular mechanisms of aluminum toxicity and resistance in plants

    Annu. Rev. Plant Physiol. Mol. Biol.

    (1995)
  • L.V. Kochian et al.

    How do crop plants tolerant acid soils? Mechanisms of aluminum toxicity and phosphorus efficiency

    Annu. Rev. Plant Biol.

    (2004)
  • L.V. Kochian et al.

    Plant adaptation to acid soils: the molecular basis for crop aluminum resistance

    Annu. Rev. Plant Biol.

    (2015)
  • I. Letunic et al.

    Interactive Tree of Life (iTOL) v4: recent updates and new developments

    Nucleic Acids Res.

    (2019)
  • J.Y. Li et al.

    Natural variation underlies alterations in Nramp aluminum transporter (NRAT1) expression and function that play a key role in rice aluminum tolerance

    Proc. Natl. Acad. Sci. U.S.A.

    (2014)
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