Silencing the Oryza sativa plasma membrane H+-ATPase isoform OsA2 affects grain yield and shoot growth and decreases nitrogen concentration
Introduction
Rice (Oryza sativa) has a high requirement for nitrogen (N), which improves the nutritional quality of grain protein (Souza et al., 1998). Fertilizing with N increases panicle numbers, spikelet numbers, grain weight, and panicle size (Fageria and Baligar, 2001). Plants respond to low soil N levels by increasing their N uptake efficiency, particularly in tropical soils. Two key components of nitrate (NO3−) uptake are plasma membrane (PM) H+-ATPases and NO3− transporters, which depend on N levels, N status, and feedback responses. The PM H+-ATPase isoforms OsA2 and OsA7 and the NO3− transporters OsNRT1.1, OsNRT2.1, and OsNRT2.2 are inducible by NO3− (Araki and Hasegawa, 2006; Sperandio et al., 2011). The NRT1.1 transporter in Arabidopsis is involved in NO3− uptake at different external NO3− concentrations (Ho et al., 2009).
PM H+-ATPases are regulated both transcriptionally and post-translationally (Falhof et al., 2016), and nutrients such as NO3−, ammonium (NH4+), and iron (III) (Fe+3) increase PM H+-ATPase transcriptional regulation and activity (Quaggiotti et al., 2003; Santi et al., 2003, 2009; Sperandio et al., 2011). In addition, low phosphorus (P) levels increase AHA2 and AHA7 expression in Arabidopsis (Yuan et al., 2017). There are 10 PM H+-ATPase isoforms in rice (OsA1–OsA10), and OsA2 and OsA7 are induced by NO3− in rice roots (Sperandio et al., 2011). Nutrient uptake depends on PM H+-ATPase activity to generate an electrochemical proton gradient (ΔμH+) and proton motive force (Δp) across the apoplast and plasma membrane (Glass, 2003; Gaxiola et al., 2007; Falhof et al., 2016). Therefore, it is necessary to understand the functions of specific PM H+-ATPase isoforms that are related to net flux of NO3− to improve the nutrient content of plants.
PM H+-ATPases are also involved in cellular growth, sugar transport, mineral nutrient translocation, grain filling, etc. (Falhof et al., 2016). PM H+-ATPase isoform characterization is well advanced in the model plant species Arabidopsis, which has 11 isoforms (Arango et al., 2003). In Arabidopsis, AHA10 is expressed mainly during seed development; AHA4 is expressed in the root endoderm, flowers, and siliques; and AHA6, AHA8, and AHA9 are expressed in pollen (Gaxiola et al., 2007). The AHA2 isoform is induced by the NO3− supply, particularly at low NO3− levels (Młodzińska et al., 2015). Młodzińska et al. (2015) showed that root systems are altered by NO3−, but the signal transduction with respect to NO3−-induced PM H+-ATPase expression and activity is not fully known. Moreover, alkaline solutions (pH 8.0) inhibit the root growth and PM H+-ATPase activity of rice, the process of which is mediated by ethylene (Chen et al., 2017).
In Arabidopsis, AHA2 and AHA7 are related to Fe uptake (Santi and Schmidt, 2009), as is CsHA1 in cucumber (Cucumis sativus) (Santi et al., 2005). In rice, OsA8 is related to soil P uptake and root-to-shoot translocation (Chang et al., 2009), and PM H+-ATPase isoforms are differentially induced by N. NO3− resupply strongly increases the expression of OsA2, while OsA5, OsA7, and OsA8 exhibit moderate increases in expression. Similarly, NH4+ resupply increases OsA1, OsA3, and OsA7 expression (Sperandio et al., 2011). PM H+-ATPase expression is related to NO3− uptake in plants. The maize (Zea mays) MHA1 isoform is more highly expressed in genotypes with high N uptake efficiency (Quaggiotti et al., 2003). Both coumarin (secondary metabolite) and NO3− increase PM H+-ATPase activity, ZmHA4 expression, and NO3− uptake in maize (Lupini et al., 2018). Moreover, copper toxicity affects NO3− uptake by downregulating low-affinity NO3− transporters and PM H+-ATPase expression (Hippler et al., 2018).
In this study, we used artificial microRNA (amiRNA) technology to investigate the function of the PM H+-ATPase OsA2 in the concentration of N in rice. Since overexpression of PM H+-ATPase may not increase its activity due to post-translational modification (Gévaudant et al., 2007; Bobik et al., 2010), the OsA2 gene was downregulated via amiRNA. Our objectives were to obtain transgenic rice that present low OsA2 expression levels by the use of a specific amiRNA (osa2), evaluate the impact of decreased OsA2 expression on rice growth and production in the greenhouse, evaluate PM H+-ATPase activity in osa2 lines, evaluate the net flux of NO3− and determine whether OsA2 expression alters expression of high-affinity NO3− transporters expression at different N levels.
Section snippets
Construction of amiRNA vectors to downregulate OsA2
AmiRNAs were designed using the website http://wmd3.weigelworld.org/cgi-bin/webapp.cgi. 5′-TCAATCTTTAACGGGTCACCT-3′ and 5′-TATCTTTAACGGATCACCCTG-3′ amiRNA sequences targeting OsA2 were designed and were named OsA2mi1 and OsA2mi2, respectively. The primers used for polymerase chain reaction (PCR) for OsA2mi1 and OsA2mi2 construction are listed in Table 1.
PCR was performed according to the methods of Warthmann et al. (2008) using a pNW55 vector containing osa-MIR528 as a template. The binary
Evaluation of osa2 lines and effects of OsA2 silencing on growth and yield
Analysis of OsA2 expression revealed that only OsA2mi2 was effective at downregulating OsA2 levels. The IRS line (control) did not significantly differ from the WT plants (Fig. 1). OsA2mi1 was not effective at downregulating its target, since OsA2 expression in the OsA2mi1 lines was not significantly reduced compared to that in the IRS control plants without amiRNA (Fig. 1). The OsA2mi2 lines L2, L3, and L4 had 80 % lower OsA2 levels than did the WT plants, and L6, L7, L8, and L9 had 64 % lower
Discussion
To determine the importance of the PM H+-ATPase OsA2 isoform in terms of N concentration, rice plants were transformed with an amiRNA targeting OsA2. Initially, two amiRNA sequences (OsA2mi1 and OsA2mi2) were used to downregulate OsA2 transcripts. Real-time PCR revealed that only OsA2mi2 was able to downregulate OsA2, highlighting the importance of using at least two amiRNAs to mediate gene silencing, as amiRNAs have been shown to have different gene silencing efficiencies (Warthmann et al.,
Author contributions
Sperandio, MVL: developing the hypothesis, constructing the amiRNA vector, producing the transgenic rice, performing the experiments and analysing the data, and writing the manuscript.
Santos, LA: developing the hypothesis, constructing the amiRNA vector, writing the Results and Discussion.
Tavares, OCH: cultivating the rice, performing the experiments, and analysing the data.
Fernandes, MS: developing the hypothesis and writing the Results and Discussion.
Lima, MF: constructing the amiRNA vector
Financial support
This study was supported by theConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
CRediT authorship contribution statement
Marcus Vinícius Loss Sperandio: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization. Leandro Azevedo Santos: Conceptualization, Investigation, Writing - original draft. Orlando Carlos Huertas Tavares: Methodology, Formal analysis. Manlio Silvestre Fernandes: Conceptualization, Writing - original draft, Funding acquisition. Marcelo de Freitas Lima: Methodology, Writing - original draft. Sonia Regina de
Declaration of Competing Interest
None.
Acknowledgements
We would like to thank Emmanuel Guiderdoni and Martine Bes (Centre de coopération internationale en recherche agronomique pour le développement (CIRAD), France) for providing the IRS154 vector and Plant Nutrition Laboratory students at UFRRJ (Federal Rural University of Rio de Janeiro, Brazil) for assisting with the harvest. We would also like to thank the CAPES, CNPq, and FAPERJ for financial support.
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In memoriam.