Plant Physiology Preview. Published on January 23, 2015, as DOI:10.1104/pp.114.251314
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Running title: FaSnRK2.6 regulates strawberry fruit ripening
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To whom correspondence should be addressed:
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Wensuo Jia, College of Agronomy and Biotechnology, China Agricultural University,
Beijing 100193, China. Email: [email protected]. Phone: 86-10-6273-1415,
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Bingbing Li, College of Agronomy and Biotechnology, China Agricultural
University, Beijing 100193, China.Email: [email protected]. Phone: 86-106273-2945;
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Research area: Genes, Development and Evolution
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Secondary Research Area: Signaling and Response
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FaSnRK2.6, an ortholog of Open Stomata 1, is a Negative Regulator of Strawberry
Fruit Development and Ripening
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Yu Han, Ruihong Dang, Jinxi Li, Jinzhu Jiang, Ning Zhang, Meiru Jia, Lingzhi Wei,
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Ziqiang Li, Bingbing Li* and Wensuo Jia*
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College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193,
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China
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*
Author for correspondence: Wensuo Jia, E-mail: [email protected], Tel: +86 10
62731415; Bingbing Li, E-mail: [email protected], Tel:+86 10 62732945
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One-sentence summary: FaSnRK2.6 is a negative regulator of strawberry fruit
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development and ripening and mediates temperature-modulated strawberry fruit
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ripening.
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ABSTRACT
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Whereas the regulatory mechanisms that direct fruit ripening have been extensively
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studied, little is known about the signaling mechanisms underlying this process
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especially for non-climateric fruits. In the present study, we demonstrated that a
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Sucrose non-fermenting 1-related protein kinase 2, designated as FaSnRK2.6, is a
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negative regulator of fruit ripening in the non-climacteric fruit Fragaria × ananassa
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(strawberry). FaSnRK2.6 was identified as an ortholog of Open Stomata 1. Levels of
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FaSnRK2.6 transcript rapidly decreased during strawberry fruit development and
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ripening. FaSnRK2.6 was found to be capable of physically interacting with FaABI1,
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a negative regulator in strawberry fruit ripening. RNA interference-induced silencing
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of FaSnRK2.6 significantly promoted fruit ripening. By contrast, over-expression of
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FaSnRK2.6 arrested fruit ripening. Strawberry fruit ripening is highly sensitive to
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temperature, with high temperatures promoting ripening and low temperatures
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delaying it. As the temperature increased, the level of FaSnRK2.6 expression declined.
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Furthermore, manipulating the level of FaSnRK2.6 expression altered the expression
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of a variety of temperature-responsive genes. Taken together, this study demonstrates
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that FaSnRK2.6 is a negative regulator of strawberry fruit development and ripening,
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and furthermore that FaSnRK2.6 mediates temperature-modulated strawberry fruit
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ripening.
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INTRODUCTION
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Ripening of fleshy fruits is a complex process that involves dramatic changes in
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colour, texture, flavour, and aroma (Giovannoni, 2001; Seymour et al., 2013). Fleshy
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fruits are physiologically classified as climacteric or non-climacteric. Whereas
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climacteric fruits exhibit an increase in respiration and a concomitant burst in ethylene
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production soon after the onset of ripening, non-climacteric fruits do not (Nitsch,
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1953; Brady, 1987; Coombe, 1976). Much of our knowledge of fruit ripening is based
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on climacteric fruits, such as tomato (Klee and Giovannoni, 2011). However, the
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mechanism underlying the ripening of non-climacteric fruits, such as strawberry, is
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poorly understood.
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Early theories on ripening emphasize the senescence-associated deteriorative
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aspects of this process; however, it is now widely accepted that ripening is a highly
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coordinated process determined by genetic programs (Brady, 1987; Fischer and
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Bennett, 1991; Giovannoni, 2001). Although the mechanisms underlying the
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molecular regulation of fruit ripening have been extensively studied, these studies
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have largely focused on the contribution of phytohormones and transcription factors
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to this process, and little is known about the signaling mechanisms that underlie fruit
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development and ripening (Giovannoni, 2001; Klee and Giovannoni, 2011; Fischer
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and Bennett, 1991; Seymour et al., 2013).
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Protein kinase/phosphatase-catalyzed reversible phosphorylation plays a central
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role in cellular signal transduction (Bourret et al., 1991; Freeman and Gurdon, 2002).
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Sucrose non-fermenting-1-related protein kinases 2 (SnRK2s) are a family of plant-
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specific protein kinases implicated in stress and ABA signaling (Yoshida et al., 2002;
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Hrabak, 2013; Kobayashi, 2004; Boudsocq, 2004; Umezawa, 2004; Fujii, 2007;
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Shukla and Mattoo, 2008; Fujita, 2009; Hirayama and Umezawa, 2010; Kulik et al.,
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2011). OST1 (Open Stomata 1, also designated as SnRK2E or SnRK2.6) is a member
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of the SnRK2 family that plays a critical role in stomatal movement, and in
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Arabidopsis, mutation of OST1 renders guard cells insensitive to ABA, such that
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stomata remain open in the presence of ABA (Mustilli et al., 2002; Assmann,
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2003;Yoshida et al., 2006; Imes et al., 2013). OST1 and its orthologs in other plant
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species are thought to have important roles in various biological processes, in addition
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to their involvement in stomatal regulation (Vilela et al., 2013; Sirichandra et al.,
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2010; Zheng et al., 2010; Xue et al., 2011; Wang et al., 2013; Imes et al., 2013).
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Recently, ABA was shown to act as an internal cue that plays an important role in
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strawberry fruit ripening (Jia et al., 2011). Furthermore, it was reported that
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manipulating the expression of either an ABA receptor, FaPYR1, or its downstream
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signaling component, FaABI1, affects strawberry fruit ripening (Chai et al., 2011; Jia
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et al., 2013). Given that OST1 is a critical mediator of ABA signaling, it is possible
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that the strawberry ortholog of OST1 plays an important role in strawberry fruit
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development and ripening. However, this possibility has not hitherto been explored.
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Fleshy fruit ripening is determined both by internal regulators, such as
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phytohormones and signaling proteins, and external environmental cues (Ulrich, 1958;
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Yang et al., 2012, Llop-Tous et al., 2002; Seymour et al., 2013; Giovannoni, 2001).
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Temperature, photon flux density, and water availability affect both fruit ripening and
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quality, and temperature is particularly important for controlling the progression of
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fruit development and ripening during fruit production. While much attention has
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focused on the mechanisms by which internal cues regulate fruit ripening, little is
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known about the mechanisms by which external cues regulate fruit development and
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ripening. Given that fleshy fruit arose as a mechanism to facilitate seed dispersal and
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promote survival of the plant in ever-changing environments (Coombe, 1976), the
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regulation of fruit ripening by external cues is of great importance in the plant’s
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adaptation to environmental stresses. Given that OST1 is a critical mediator of ABA
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signaling, and that SnRK2s are tightly associated with plant stress signaling (Yoshida
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et al., 2002; Hrabak, 2013; Kobayashi, 2004; Boudsocq, 2004; Umezawa, 2004; Fujii,
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2007; Shukla and Mattoo, 2008; Fujita, 2009; Hirayama and Umezawa, 2010; Kulik
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et al., 2011), we sought to identify the ortholog of OST1 in strawberry and to evaluate
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whether this protein contributes to the regulation of strawberry fruit development and
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ripening, and particularly whether it modulates fruit ripening in response to
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temperature.
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RESULTS
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Genome-wide identification of FaSnRK2 subfamily members
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To identify the strawberry ortholog of OST1, we first searched for Sucrose non-
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fermenting-1 related protein kinase 2 (SnRK2s) subfamily members in the strawberry
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genome.
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(https://strawberry.plantandfood.co.nz/) using the coding sequences (CDSs) of all
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members of the Arabidopsis thaliana SnRK subfamily as query (Boudsocq et al.,
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2004) detected nine non-redundant gene sequences. Protein sequence alignment
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showed that all nine members contained a conserved SnRK2 domain (Hrabak et al.,
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2003; Boudsocq et al., 2004), and we therefore named these proteins FaSnRK2.1 to
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FaSnRK2.9, respectively (Fig. 1A). To compare FaSnRK2s with AtSnRK2s and other
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SnRK2s previously reported (Park Y S et al., 1993; Mustilli A C et al., 2002; Hrabak
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E M et al., 2003; Mao X et al., 2010; Bucholc M et al., 2011), we constructed a
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phylogenetic tree based on the full-length cDNA sequences of the selected SnRK2s
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using the neighbor-joining (N-J) method in MEGA6 software (Fig. 1B). The
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FaSnRK2s clustered into four clades. Among all of the sequences selected,
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OST1/AtSnRK2.6 shares the highest degree of similarity (95%) with FaSnRK2.6.
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Within the FaSnRK2s subfamily, FaSnRK2.6 and FaSnRK2.3 belong to the same
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clade, and share only 80% sequence similarity. The high degree of similarity between
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FaSnRK2.6 and OST1 suggests that FaSnRK2.6 is an ortholog of OST1.
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Temporospatial expression of FaSnRK2.6
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Using quantitative PCR, we showed that FaSnRK2.6 was ubiquitously expressed in
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strawberry plants, with the highest transcript level detected in the leaves (Fig. 2A). We
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were particularly interested in determining the expression pattern of FaSnRK2.6
BLAST
searches
against
the
strawberry
sequence
database
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during fruit development and ripening. The process of strawberry fruit development,
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from fruit set to ripening, can be divided into three major stages, i.e., the green fruit
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stage, white fruit stage (W), and red fruit stage, and the green and red fruit stages can
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again be divided into several substages, i.e., the small green fruit (SG), middle green
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fruit (MG), and large green fruit (LG) and initially reddening (IR) and fully reddening
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(FD) substages (Fig. 2B). As shown in Fig. 2C, FaSnRK2.6 was strongly expressed in
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the SG substage, declined sharply from the MG to LG substages, and then decreased
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gradually until the FD substage, suggesting that there is a tight negative correlation
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between FaSnRK2.6 expression and the progression of fruit development and ripening.
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As a comparison, we also examined the expression patterns of other members of the
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FaSnRK2 subfamily. While the expression of FaSnRK2.3 and FaSnRK2.5 also
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declined throughout fruit growth and development, no clear correlations were detected
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between the expression of other members and fruit ripening (Fig. 2C).
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Expression of FaSnRK2.6 in response to internal signals
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As ABA and sucrose have been identified as internal signals that control strawberry
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fruit development and ripening (Jia et al., 2011; Jia et al., 2013), we examined the
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expression of FaSnRK2.6 in response to ABA and sucrose stimuli in fruits. As shown
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in Fig. 3A, the level of FaSnRK2.6 expression declined upon ABA treatment in a
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concentration-dependent manner. Furthermore, FaSnRK2.6 expression was sensitive
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to ABA, as just 20 mM ABA significantly reduced FaSnRK2.6 expression. Based on
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the findings that OST1 regulates stomatal movement in leaves (Mustilli et al., 2002)
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and that FaSnRK2.6 is largely expressed in leaves (Fig. 2A), we further examined the
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response of FaSnRK2.6 expression to ABA in leaves. Unexpectedly, FaSnRK2.6
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expression in the leaves was not affected by ABA treatment (Fig. 3B), suggesting that
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different mechanisms regulate FaSnRK2.6 expression in leaves and fruits.
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Additionally, sucrose treatment had no effect on the level of FaSnRK2.6 expression in
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fruits, suggesting that FaSnRK2.6 is specifically involved in ABA signaling (Fig. 3C).
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Manipulation of FaSnRK2.6 expression modulated fruit development and
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ripening
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The close relationship between the pattern of FaSnRK2.6 expression and fruit
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development and ripening (Fig. 2) suggests that FaSnRK2.6 is an important regulator
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of strawberry fruit development and ripening. To test this possibility, we performed
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loss- and gain- of function experiments using transient over-expression (OE) and
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RNA-interference (RNAi) techniques (Jia et al., 2013). As shown in Fig. 4A,
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FaSnRK2.6-OE resulted in a more than 4-fold increase in the level of FaSnRK2.6
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transcript, whereas FaSnRK2.6-RNAi resulted in a dramatic decrease in FaSnRK2.6
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transcript level. Thus, the transient OE and RNAi techniques successfully
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manipulated FaSnRK2.6 expression in the strawberry fruits. Interestingly,
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FaSnRK2.6-OE greatly delayed the progression of strawberry fruit development and
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ripening. While the control fruits transfected with empty vector became red, the fruits
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of FaSnRK2.6-OE plants commonly remained in the W stage (Fig. 4B). By contrast,
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while the control fruits were still in the W stage, the fruits of FaSnRK2.6-RNAi
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commonly became red (Fig. 4C).
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Consistent with these phenotypic observations, FaSnRK2.6-OE and -RNAi strongly
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affected ripening-related physiological and biochemical metabolism and the related
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gene expression (Table 1). Compared with the corresponding control, the FaSnRK2.6-
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OE and -RNAi lines exhibited a large increase and decrease in fruit firmness,
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respectively, and conversely, a large decrease and increase in anthocyanin content.
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FaSnRK2.6-OE and -RNAi also resulted in significant changes in major fruit quality
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parameters such as flavonoid and total phenol content. Furthermore, FaSnRK2.6-OE
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and -RNAi led to an increase and decrease, respectively, in the content of aldehyde
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and alcohol compounds (such as hexanal, 2-hexenal, octanal, etc. ), and conversely, a
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decrease and increase in the content of keton, furan, and ester compounds (such as
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4H-Pyran-4-one-2,3-dihydro-3,5-dihydroxy-6-methyl, hexanoic acid, etc.) Given that
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mature fruits contain greater concentrations of ketone, furan, and ester compounds
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and lower concentrations of aldehyde and alcohol compounds than immature fruit
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(Hadi et al., 2013), the changes in aroma, pigment, and cell wall metabolism in the
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FaSnRK2.6-OE and –RNAi lines collectively suggested that the FaSnRK2.6-OE and –
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RNAi lines inhibited and promoted strawberry fruit ripening, respectively.
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Consistent with the observations on the changed patterns of the ripening-related
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physiological parameters as described above, FaSnRK2.6-OE and -RNAi dramatically
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modulated the expression of a series of genes implicated in fruit color and aroma
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metabolism (Fig. 5), including FaCHS (chalcone synthase), FaCHI (chalcone
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isomerase), FaF3H (flavanone 3-hydroxylase), FaUFGT (UDP-glycose flavonoid 3-
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O-glycosyltransferase), Fa4CL1 (4-coumaroyl-CoA ligase 1), and Fa4CL2 (4-
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coumaroyl-CoA ligase 2), in a converse manner. Notably, FaSnRK2.6-OE almost
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completely blocked the expression of FaQR (quinine oxidoreductase), a key gene
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implicated in aroma metabolism, whereas FaSnRK2.6-RNAi resulted in a more than
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5-fold increase in the level of FaQR transcript (Fig. 5A). Also in a converse manner,
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FaSnRK2.6-OE and FaSnRK2.6-RNAi greatly affected the expression of a series of
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genes implicated in fruit softening, such as FaPE (pectinesterase), FaCEL (cellulose),
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FaGAL2 (beta-galactosidase2), FaXYL1 (beta-xylosidase1), and FaEXP1 (expansin1).
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Collectively, these results strongly indicate that FaSnRK2.6 plays a critical role in the
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regulation of strawberry fruit development and ripening.
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Subcellular localization of FaSnRK2.6 and the physical interaction between
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FaSnRK2.6 and FaABI1
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A previous study demonstrated that FaABI1, a key signal in the ABA signaling
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cascade, is a negative regulator of strawberry fruit development and ripening (Jia et
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al., 2013). We here examined the physical interaction between FaSnRK2.6 and
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FaABI1. FaSnRK2.6-GFP fluorescence indicated that FaSnRK2.6 was mainly
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localized to the nucleus and cytoplasm of Zea mays (maize) protoplasts (Fig. 6A).
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Yeast two-hybrid analysis indicated that FaSnRK2.6 undergoes auto-activation,
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because yeast cells continued to grow well on SD/-ade,-leu,-trp,-his medium when
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harboring BD-FaSnRK2.6 and AD empty plasmid, but not when harboring AD-
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FaSnRK2.6 and BD empty plasmid. The clear positive result for the combination of
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BD-FaABI1 and AD-FaSnRK2.6 indicates that FaSnRK2.6 physically interacts with
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ABI1 (Fig. 6B). To provide further evidence for the interaction between FaSnRK2.6
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and FaABI1, we performed a BiFC assay. As shown in Fig. 6C, fluorescence was
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clearly observed for both the FaABI1-YFPN and FaSnRK2.6-YFPC combination and
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the FaABI1-YFPC and FaSnRK2.6-YFPN combination. By contrast, no fluorescence
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was observed for the other combinations tested, strongly suggesting that FaSnRK2.6
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interacts with FaABI1.
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Temperature-modulated fruit development and ripening in relation to
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FaSnRK2.6 expression
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Since temperature is a major environmental cue affecting strawberry development and
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ripening, we investigated a possible role for FaSnRK2.6 in temperature-modulated
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fruit ripening. To evaluate the effect of temperature on strawberry fruit development
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and ripening, plants were first grown under normal conditions until fruit set (i.e.,
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day/night, 25/18°C), and were then divided into two groups and grown either under a
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day/night regimen of 32/20°C or 20/12°C. As shown in Fig. 7, the two different
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temperature treatments resulted in great differences in the progression of strawberry
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fruit development and ripening, with fruit grown at the higher temperature ripening
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approximately 10 days before that grown at the lower temperature, indicating that
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strawberry fruit ripening is sensitive to temperature.
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To further investigate whether FaSnRK2.6 might be associated with temperature-
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modulated fruit development and ripening, we examined the effect of temperature on
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FaSnRK2.6 expression. As shown in Fig. 8A, transient exposure to high temperature
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greatly reduced the expression of FaSnRK2.6 (Fig. 8A), and by contrast, low
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temperature treatment completely blocked the fruit development-induced reduction in
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FaSnRK2.6 expression (Fig. 8B). These results suggest that the pattern of
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temperature-modulated FaSnRK2.6 expression was consistent with that of FaSnRK2.6
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expression during the progression of strawberry fruit ripening and development.
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Together with the finding that strawberry fruit development and ripening could be
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manipulated by modulating the expression of FaSnRK2.6 (Fig. 4, Fig. 5, and Table 1),
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these results suggest that FaSnRK2.6 mediates temperature-modulated fruit
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development and ripening.
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Since FaSnRK2.6 was largely expressed in leaves (Fig. 2A), and importantly, since
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its ortholog OST1 controls stomatal movement in leaves (Mustilli et al., 2002), we
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also examined FaSnRK2.6 expression in response to temperature stimuli in leaves
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(Fig. 8C and 8D). Interestingly, in response to high temperature treatment, FaSnRK2.6
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expression initially declined but rapidly recovered to its original level (Fig. 8C).
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Moreover, in response to low temperature treatment, FaSnRK2.6 expression decreased
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rather than increased in leaves, suggesting that the mechanism underlying the
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regulation of FaSnRK2.6 expression is distinctly different in fruits and leaves.
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Roles of FaSnRK2.6 in temperature-modulated gene expression
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To provide further evidence that FaSnRK2.6 is involved in temperature-modulated
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fruit development and ripening, we examined whether manipulating FaSnRK2.6
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expression by OE or RNAi would alter the expression of temperature-responsive
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genes in fruits. To do so, we first transfected fruits at the LG stage with FaSnRK2.6-
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OE, FaSnRK2.6-RNAi, or empty vector constructs and incubated the fruits for 5 days
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at room temperature (25°C). We then subjected these fruits to a transient treatment of
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high (40°C, 8 h) or low (4°C, 24 h) temperature and tested for an expressional
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induction of typical temperature-responsive genes, such as those implicated in color
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metabolism (e.g., FaCHS, FaCHI, and FaUFGT), cell wall metabolism (e.g., FaPE
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and FaXYL1), and aroma metabolism (e.g., FaQR).
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As shown in Fig. 9A, transient transfection with FaSnRK2.6-OE and FaSnRK2.6-
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RNAi respectively resulted in a marked increase and decrease in the level of
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FaSnRK2.6 transcript. High or low temperature treatment respectively resulted in a
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significant decrease or increase in the level of FaSnRK2.6 transcript in the fruits
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transfected with the empty vector (i.e., EV), whereas it had no significant effect on
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fruits transfected with FaSnRK2.6-OE. High or low temperature also respectively
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resulted in a decrease or increase in the level of FaSnRK2.6 transcript in FaSnRK2.6-
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RNAi fruits, but to a lesser extent than in fruits transfected with the EV.
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As shown in Fig. 9B and 9C, while the over-expression or silencing of FaSnRK2.6
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respectively resulted in a large increase or decrease in the level of FaSnRK2.6
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transcript (9A), they respectively resulted in a large decrease or increase in the
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transcript levels of the ripening-related genes relative to the EV control, regardless of
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the temperature treatment applied (9B and 9C). High or low temperature respectively
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induced or suppressed the expression of ripening-related genes in fruits transfected
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with EV (Fig. 9B and 9C), but the induction or suppression of these genes was
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completely blocked in fruits transfected with FaSnRK2.6-OE. These findings suggest
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that the expression of the temperature-responsive genes can be modulated by
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manipulating the expression of FaSnRK2.6. Collectively, these results strongly
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suggest that (1) FaSnRK2.6 plays a critical role in temperature-modulated gene
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expression, and (2) the FaSnRK2.6-mediated regulation of the ripening-related genes
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in response to a temperature stimulus is largely achieved by regulating FaSnRK2.6 at
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the transcriptional level.
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Methylation analysis of the FaSnRK2.6 promoter
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DNA methylation is a key mechanism by which the expression of a specific gene is
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regulated in different cells/tissues (Finnegan et al., 1998). Given that strawberry fruit
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development and ripening are largely controlled by the transcriptional regulation of
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FaSnRK2.6 and that temperature had a different effect on FaSnRK2.6 expression in
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fruits and leaves, we analyzed the DNA methylation status of the FaSnRK2.6
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promoter in relation to its expression in fruits and leaves. The fragment 2288 bp
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upstream of the FaSnRK2.6 transcriptional start site was isolated and demonstrated to
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have promoter activity in a GUS-mediated gene expression analysis (Fig. 10A). As
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expected, many sites within this fragment were differently methylated in DNA
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isolated from fruits and leaves. Under the control temperature (25°C), no methylation
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occurred at positions -1942, -1852, -1846, and -1136 bp relative to the transcriptional
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start site of FaSnRK2.6 in DNA isolated from fruits, whereas all of these sites were
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methylated in DNA from leaves. Interestingly, while high temperature treatment
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resulted in methylation at positions -1852, -1846, -1839, and -1109 bp of FaSnRK2.6
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relative to the transcriptional start site and de-methylation at position -1912 bp in
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DNA isolated from fruits, it resulted in de-methylation at positions -1852 and -1846
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bp and methylation at position -1140 bp in leaves. Also, while low temperature
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treatment resulted in methylation at position -1136 and -1117 bp and de-methylation
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at position -1912 bp of FaSnRK2.6 in DNA isolated from fruits, it resulted in
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methylation at positions -1754, -1140, and -1109 bp and de-methylation at position -
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1852 and -1846 bp in that isolated from leaves (Fig. 10B), suggesting that both high
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and low temperature have different effects on the methylation and de-methylation of
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DNA isolated from fruit and leaves. Bioinformation analysis indicated that most of
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the sites involved in the regulation of DNA methylation were within the cis-elements
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of the FaSnRK2.6 promoter and that these sites had a variety of different functions
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(see Supplemental Table 7). Collectively, these results suggest that modification of
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promoter methylation is an important mechanism by which FaSnRK2. 6 expression is
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uniquely regulated in fruits and leaves.
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DISCUSSION
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Previous studies demonstrated that ABA as well as PYR1 and FaABI1 were involved
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in the regulation of strawberry fruit development and ripening (Chai et al., 2011, Jia et
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al., 2011, 2013). It is not surprising that PYR1 and ABI1 are implicated in strawberry
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fruit development and ripening, because molecular interaction between PYR and
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ABI1 is required to initiate ABA signaling. In the present study, we identified a novel
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signal component, FaSnRK2.6, that functions downstream of PYR and ABI1 and
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serves as a negative regulator of fruit development and ripening. Interestingly,
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FaSnRK2.6 was the only ortholog of OST1 identified in the strawberry genome (Fig.
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1), implying that FaSnRK2.6 may regulate both stomatal movement and fruit ripening.
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Given that FaSnRK2.6 is a negative regulator of strawberry fruit development and
374
ripening, whereas OST1 is a positive regulator of stomatal movement, FaSnRK2.6
375
must operate via distinct mechanisms in these biological processes. Although SnRK2
376
protein kinases have been suggested to be downstream signal transducers of ABA
377
(Kobayashi et al., 2005; Wang et al., 2013) and although we found that SnRK2.6
378
physically interacts with FaABI1 (Fig. 6), the FaSnRK2.6-mediated regulation of
13
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
379
strawberry fruit development and ripening does not likely occur via the post-
380
transcriptional regulation of FaSnRK2.6 by the ABA/FaPYR1/FaABI1 signaling
381
cascade, because this signaling cascade would be expected to activate rather than de-
382
active FaSnRK2.6, based on our understanding of ABA signaling (Soon et al., 2012;
383
Wang et al., 2013). The most likely mechanism by which FaSnRK2.6 mediates ABA-
384
regulated strawberry fruit ripening is the regulation of FaSnRK2.6 at the
385
transcriptional level. Consistent with this notion, we found that FaSnRK2.6
386
expression could be sensitively inhibited by ABA in a concentration-dependent
387
manner (Fig. 3A). These results, in combination with the previous observations that
388
ABA content is substantially increased during strawberry fruit development and
389
ripening (Jia et al., 2011), strongly suggest that FaSnRK2.6 mediates ABA-regulated
390
fruit ripening mainly through the transcriptional regulation of FaSnRK2.6.
391
392
Fruit ripening is not only regulated by internal signals, but is also affected by a
393
variety of environmental factors, such as light, water availability, and temperature
394
(Ulrich, 1958; Yang et al., 2012; Llop-Tous et al., 2002; Seymour et al., 2013;
395
Giovannoni, 2001). In the present study, we found that strawberry fruit development
396
and ripening are indeed very sensitive to changes in temperature (Fig. 7). Consistent
397
with the observation that high temperature dramatically accelerated the onset of fruit
398
ripening (Fig. 7), we found that the expression of the ripening-related genes could be
399
sensitively regulated by temperature, i.e., the expression of a series of ripening-related
400
genes was rapidly upregulated upon exposure to a high temperature stimulus (Fig. 9B).
401
By contrast, the expression of FaSnRK2.6 rapidly decreased upon exposure to a high
402
temperature stimulus (Fig. 8A). These results suggest that the temperature-modulated
403
expression of ripening-related genes is correlated with the regulation of FaSnRK2.6
404
expression. Importantly, we found that over-expression of FaSnRK2.6 completely
405
blocked high temperature-induced gene expression, which suggests that the
406
temperature-modulated expression of ripening-related genes is mediated by
407
FaSnRK2.6. Furthermore, the transcript levels of ripening-related genes were always
408
negatively correlated with the transcript level of FaSnRK2.6, regardless of the
14
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
409
temperature treatment applied (Fig 9B and 9C), implying that the temperature-
410
modulated expression of the ripening-related genes also largely occurs through the
411
transcriptional regulation of FaSnRK2.6.
412
413
The SnRK2s family has been suggested to have evolved specifically for
414
hyperosmotic stress signaling (Kobayashi et al., 2004; Boudsocq et al., 2004;
415
Umezawa et al., 2004; Shukla and Mattoo 2008; Fujita et al., 2009; Fujii et al., 2011).
416
For example, Kobayashi et al. (2004) identified 10 SnRK2 protein kinases encoded in
417
rice (Oryza sativa), and all of these SnRK2s were specifically activated by osmotic
418
stress at both the transcriptional and the post-transcriptional level. The involvement of
419
the stress signal, FaSnRK2.6, in the regulation of fruit development and ripening may
420
reasonably be explained from a plant ecological perspective. Seeds are known to be
421
the most stress-tolerant organ, and hence, facilitating seed dispersal can be regarded
422
as a strategy for plants to cope with stresses. From an evolutionary perspective, fleshy
423
fruits arose to facilitate seed dispersal, thereby enabling plants to survive and thrive in
424
ever-changing environments (Nitsch, 1953; Brady, 1987; Coombe, 1976).
425
Accordingly, the involvement of FaSnRK2.6 in the regulation of fruit development
426
and ripening may just be a consequence of its essential role in plant stress signaling.
427
While mechanisms of plant stress signaling have been extensively studied, much less
428
is known about the signaling mechanisms underlying fruit development and ripening.
429
Knowledge of plant stress signaling may provide important clues for better
430
understanding the molecular mechanisms underlying strawberry fruit development
431
and ripening.
432
433
Ripening of fleshy fruits has been generally thought of as a distinct developmental
434
stage, during which dramatic changes in a series of physiological and biochemical
435
metabolic processes occur, including modification of cell wall ultrastructure and
436
texture, increases/decreases in the levels of sugars/organic acids, and the biosynthesis
437
and accumulation of aromatic volatiles (Seymour et al., 1993; Giovannoni, 2001;
438
Seymour et al., 2013). A key question regarding the ripening process is how an
15
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
439
assortment of otherwise unrelated pathways and processes act coordinately during this
440
stage of fruit development (Giovannoni, 2001). Basic cell biology studies have
441
established that in both eukaryotic and prokaryotic organisms, coordinated cellular
442
metabolism is essentially based on the regulation of cellular signaling (Anantharaman
443
et al., 2007; Bourret et al., 1991; Freeman and Gurdon, 2002; Michael et al., 2005;
444
Emily, 2012). Given the complexity of the metabolic processes occurring during fruit
445
ripening, it is not difficult to imagine that fruit ripening must be tightly controlled
446
through a complicated signaling network. In the present study, we have found that
447
suppression of FaSnRK2.6 accelerated the biosynthesis of anthocyanins, flavonoids,
448
and total phenols and reduced fruit firmness, whereas it appeared to have no
449
significant effect on the sugar and total titratable acid contents. It is likely that for
450
each metabolic pathway there exists a distinct signaling cascade, such that a single
451
signaling cascade or component is not enough to govern all of the ripening-related
452
metabolic processes. Alternatively, FaSnRK2.6 alone may not be capable of
453
controlling all of the ripening-related metabolic pathways, which implies that the
454
different pathways of the ripening-related metabolic processes are temporospatially
455
separated.
456
In summary (Fig. 11), we have identified a novel protein signal, FaSnRK2.6, that
457
regulates strawberry fruit development and ripening. FaSnRK2.6 is a downstream
458
signal of ABA, as indicated by its physical interaction with FaABI1. FaSnRK2.6
459
expression is not only negatively correlated with strawberry fruit development and
460
ripening, but is also negatively regulated by ABA and temperature, internal and
461
external cues, respectively, that are known to modulate strawberry fruit development
462
and ripening. Manipulating FaSnRK2.6 expression was capable of modulating
463
strawberry fruit ripening as well as the expression of a variety of ripening-related
464
genes, and moreover it was also capable of modulating the expression of a variety of
465
temperature-responsive genes. Collectively, these results demonstrate that FaSnRK2.6
466
is an important regulator of strawberry fruit development and ripening. The
467
FaSnRK2.6-mediated signaling pathway implicated here in temperature-modulated
468
fruit ripening embodies a wonderful strategy by which FaSnRK2.6 functions as a
16
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
469
stress signal, from a plant ecological perspective. This study greatly enhances our
470
understanding of the regulatory mechanism underlying the ripening of non-climacteric
471
fruits.
472
473
MATERIALS AND METHODS
474
Plant materials and growth conditions
475
Strawberry plants (Fragaria x ananassa Ducherne, cv Benihoppe) were planted in
476
pots (diameter, 230 mm; depth, 230 mm) containing a mixture of nutrient soil,
477
vermiculite, and organic fertilizer (7:2:1, v/v/v). The seedlings were grown in a
478
controlled environment with the following conditions: 25°C:18°C (day:night), 60%
479
humidity, and a 12 h photoperiod with a photosynthetic photon flux density (PPFD) of
480
450 µmol m-2s-1.
481
Gene isolation and analysis
482
Total RNA was extracted from fruit receptacles using an SV Total RNA Isolation
483
System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
484
To identify SnRK2s from Fragaria x ananassa, the coding sequences (CDSs) of all
485
members of the AtSnRK2 subfamily in Arabidopsis were BLASTed in the website
486
https://strawberry.plantandfood.co.nz/. Nine SNF1-related protein kinases, which
487
were designated as FaSnRK2.1~FaSnRK2.9, respectively, were identified. The
488
FaSnRK2s were cloned from cDNA by PCR under the following conditions: 98°C for
489
30 s, followed by 35 cycles of 98°C for 10 s, 54°C for 20 s, and 72°C for 2 min, with
490
a final extension of 72°C for an additional 5 min (Q5 High-Fidelity DNA Polymerase,
491
NEB). The PCR products were ligated into a pMD19-T simple vector and
492
subsequently transformed into Escherichia coli DH5α. Positive colonies were selected
493
and amplified, and then sequenced by Invitrogen. The nine FaSnRK2 sequences were
494
submitted to the NCBI database (http://www.ncbi.nlm.nih.gov/genbank/). Primer
495
sequences and GenBank accession numbers are shown in Supporting Information
496
Table 1.
17
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
497
The deduced amino acid sequences of FaSnRK2.1-FaSnRK2.9 were aligned using
498
Clustal X 2.0.12 with default settings. The alignment results were edited and marked
499
using GeneDoc.
500
Phylogenetic analysis
501
The full-length SnRK2 cDNA sequences from Arabidopsis thaliana, Zea mays,
502
Triticum polonicum L, Solanum tuberosum, Triticum aestivum, and Nicotiana
503
plumbaginifolia
504
(http://www.ncbi.nlm.nih.gov/ nucleotide/ ) as reported in previous studies (Park et al.,
505
1993; Mustilli et al., 2002; Hrabak et al., 2003; Mao et al., 2010; Bucholc et al.,
506
2011).
were
obtained
from
the
NCBI
nucleotide
database
507
The DNA sequences of FaSnRKs were aligned with the homologous genes in
508
Arabidopsis , Z. mays, Triticum polonicum L, Solanum tuberosum, Triticum aestivum,
509
and Nicotiana plumbaginifolia using ClustalX 2.0.12 software with default settings.
510
The phylogenetic trees were constructed using the neighbor-joining (N-J) method in
511
MEGA6 software with 1000 bootstrap replicates for evaluating the reliability of
512
different phylogenetic groups. Tree files were viewed and edited using MEGA6
513
software.
514
Genes and accession numbers are as follows: AtSnRK2.1, NM120946; AtSnRK2.2,
515
AT3G50500.2; AtSnRK2.3, NM126087; AtSnRK2.4, AT1G10940.2; AtSnRK2.5,
516
NM125760;
517
AT1G78290.2; AtSnRK2.9, NM127867; AtSnRK2.10, NM104774; ZmSnRK2.1,
518
EU676033;
ZmSnRK2.2,
EU676034;
ZmSnRK2.3,
EU676035;
ZmSnRK2.4,
519
EU676036;
ZmSnRK2.5,
EU676037;
ZmSnRK2.6,
EU676038;
ZmSnRK2.7,
520
EU676039; ZmSnRK2.8, EU676040; ZmSnRK2.10, EU676041; ZmSnRK2.11,
521
EU676042; TpSnRK2.2, KF688097; TpSnRK2.5, KF688098; TpSnRK2.10, KF688099;
522
TpSnRK2.11,
523
StSnRK2.3,JX280913; StSnRK2.4, JX280914; StSnRK2.5, JX280915; StSnRK2.6,
524
JX280916; StSnRK2.7, JX280917; StSnRK2.8, JX280918; TaSnRK2.4, GQ384359;
AtSnRK2.6,
KF688100;
NM119556;
StSnRK2.1,
AtSnRK2.7,
JX280911;
NM120165;
StSnRK2.2,
AtSnRK2.8,
JX280912;
18
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
525
and NtSnRK2, FJ882981.
526
Temporospatial expression of FaSnRK2.6
527
Fruit ripening stages were divided according to days post anthesis (designated
528
hereafter as DPA) and fruit development, as follows: small green fruit (SG; 6 DPA);
529
middle green fruit (MG; 12 DPA); large green fruit (LG; 18 DPA); white fruit stage
530
(W; 24 DPA); initially reddening (IR; 27 DPA); and fully reddening (FR; 30 DPA).
531
Ten fruits were harvested at each stage, and the root, stem, leaf, and flower used for
532
organ-specific expression analysis were sampled from ten plants grown under the
533
same conditions. All fruits (without seeds) and samples were immediately frozen in
534
liquid nitrogen, mixed, and stored at –80°C until further use. qPCR primers to detect
535
FaSnRK2.1~FaSnRK2.9
536
(http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi). Primer sequences are
537
shown in Supporting Information Table 2.
538
qRT-PCR analysis
539
Total RNA was extracted from fruits using an SV Total RNA Isolation System
540
(Promega, Madison, WI, USA) according to the manufacturer’s instructions. To
541
eliminate genomic DNA contamination, RNA was treated with DNase I (Takara) for
542
20 min. First-strand cDNA was synthesized from total RNA with the Takara RNA
543
PCR Kit (Takara). qRT-PCR was performed in the ABI 7500 Real-Time PCR System
544
(Applied Biosystems) following the manufacturer’s instructions and using SYBR
545
Premix ExTaq (Perfect Real Time; Takara Bio Inc., Otsu, Japan). qRT-PCR was
546
conducted with three biological replicates, and each sample was analyzed at least in
547
triplicate and normalized using FaACTIN as an internal control. Transcription levels
548
are presented in the form 2–ΔΔCT, where ΔCT represents the difference between the CT
549
values of the target and the reference genes (Thomas D et al., 2008). Primers used for
550
the qRT-PCR analysis of ripening-related genes are provided in Supporting
551
Information Table 3.
552
ABA and sucrose treatment
were
designed
using
Primer3Plus
19
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
553
Strawberry fruit and leaves were incubated in vitro with ABA as described (Beruter et
554
al., 1995; Jiang et al., 2001). After washing with distilled water, the freshly harvested
555
fruits or leaves were dried carefully. Discs (10 mm in diameter and 1 mm in thickness)
556
of fruit (with seeds removed) or leaves were prepared. The disc samples were first
557
vacuum-infiltrated for 30 minutes in equilibration buffer (Archbold D D et al., 1999)
558
consisting of 50 mM MES-Tris (pH 5.5), 10 mM MgCl2, 10 mM EDTA, 5 mM CaCl2,
559
200 mM mannitol, and 5 mM vitamin C, and then shaken for 1 or 6 h at 25°C in
560
equilibration buffer contraning various concentrations of ABA (Sigma A1049) or
561
sucrose. After incubation, the samples were washed with double distilled water,
562
frozen immediately in liquid nitrogen, and kept at -80°C until further use. These
563
experiments were repeated three times.
564
Construction of FaSnRK2.6 over-expression and RNA interference plasmids
565
To generate the FaSnRK2.6 over-expression construct, full-length FaSnRK2.6 cDNA
566
was obtained by reverse transcription-polymerase chain reaction (RT-PCR) using the
567
following primers: forward, 5’- GTCGACATGGATCGGTCTATGCT-3’ (with a SalI
568
restriction site); reverse, 5’-AAGCTTTCAAATTGCATACACTATTTCC-3’ (with a
569
HindIII restriction site). The cDNA was cloned into the SalI and HindIII restriction
570
sites of the plant expression vector pCambia1304. To generate an intron containing a
571
hairpin RNA interference (RNAi) fragment of FaSnRK2.6, a 720-bp fragment near the
572
5’ end of FaSnRK2.6 cDNA was PCR amplified using the primer pair 5’-
573
GCATGCATGGATCGGTCTATGCTG-3’ (with a SphI restriction site; forward) and
574
5’-GTCGACGTACTGGACACTCGTTATACGATGT-3’(with a SalI restriction site;
575
reverse). The 720-bp fragment obtained was sequenced and forward cloned into the
576
SphI and SalI restriction sites of pCambia1304. A 281-bp fragment complementary to
577
the 3’end of the 720-bp fragment described above was PCR amplified from
578
strawberry
579
GCATGCACACATTGTTAGATGGAAGTC-3’ (with a SphI restriction site; forward)
580
and 5’-AAGCTTGTACTGGACACTCGTTATACGATG-3’ (with a HindIII restriction
581
site; reverse). The 281-bp fragment obtained was sequenced and forward cloned into
fruit
cDNA
using
the
following
primers:
5’-
20
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
582
the SphI and HindIII restriction sites of pCambia1304; by this means, a hairpin would
583
be produced when this 281-bp fragment was exposed to its complementary sequence
584
at the 3’end of the 720-bp fragment.
585
586
Transfection of strawberry by agroinfiltration
587
Agrobacterium tumefaciens strain AGL0 (Lazo et al., 1991), containing pCambia1304,
588
OE-pCambia1304-FaSnRK2.6, or RNAi-pCambia1304-FaSnRK2.6 was grown at
589
28°C in Luria Bertani (LB) liquid medium containing 10 mM MES and 20 µM
590
acetosyringone with appropriate antibiotics. When the culture reached an optical
591
density at 600 nm (OD600) of c.1.0, Agrobacterium cells were harvested and
592
resuspended in infection buffer (10 mM MgCl2, 10 mM MES, pH 5.6, 200 µM
593
acetosyringone) and shaken for 2 h at room temperature before being used for
594
infiltration. For transfection, attached fruits with basically identical sizes and shapes at
595
18 DPA were selected, and the Agrobacterium suspension was evenly injected into the
596
fruits with a syringe until the whole fruit became hygrophanous (Jia et al., 2013). The
597
transfections were carried out in different batches according to different expermental
598
aims, and in each batch, 40 fruits were injected, with 20 fruits being transformed with
599
FaSnRK2.6-RNAi or FaSnRK2.6-OE and another 20 used as the control (i.e., injected
600
with the pCambia1304 empty vector). To examine the effect of FaSnRK2.6-RNAi and
601
FaSnRK2.6-OE on the expression of ripening-related genes, the fruits were collected
602
7 and 12 days after the transfection, respectivley, for FaSnRK2.6-RNAi and
603
FaSnRK2.6-OE. For the expressional analyis of ripening-related genes, five fruits
604
were combined as an indivual sample. Seeds of the fruit samples were removed and
605
the receptacles were frozen in liquid nitrogen and stored at –80°C until use. To
606
examine the effect of FaSnRK2.6-RNAi and FaSnRK2.6-OE on temperature-
607
modulated gene expression, the fruits were collected 5 days after transfection, and
608
then treated with different temperature regimens as described below.
609
610
Temperature treatment experiment
611
To observe the effect of temperature on the progression of fruit development and
21
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
612
ripening, strawberry seedlings were first grown in pots until fruit set under normal
613
conditions as described in “Plant materials and growth conditions”. On the day before
614
temperature treatment started, flower and fruit thinning were performed so that for
615
each plant only 5 fruits were retained. For temperature treatment, the plants were
616
divided into two groups. One group was grown in a controlled environment under
617
32°C:20°C (day:night), 60% humidity, and a 12-h photoperiod with a photosynthetic
618
photon flux density (PPFD) of 450 µmol m-2s-1, and the other group was grown under
619
20°C:12°C (day:night), 60% humidity, and a 12-h photoperiod with a photosynthetic
620
photon flux density (PPFD) of 450 µmol m-2s-1. The numbers of mature fruits as
621
judged by changes in fruit color were successively recorded until all fruits matured.
622
To examine the effect of temperature on the expression of ripening-related genes, for
623
each treatment, 15 detached fruits of 18 DPA were used, with 5 fruits combined into
624
one sample, such that the 15 fruits were divided into three samples. The samples were
625
individually used for the analysis of gene expression. The fruits were separated
626
longitudinally into two even halves, and one half was treated with high or low
627
temperature and the other at 25°C as the control. The high temperature treatment was
628
performed at 40°C for 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, or 8 h and the low tempearture
629
treatment was performed at 4°C for 0 h, 2 h, 4 h, 8 h, 24 h, or 48 h. After the
630
temperature treatment, the seeds were removed and the fruits were frozen in liquid
631
nitrogen and stored at –80°C until further use. For temperature treatment of leaves,
632
whole plants grown in pots were treated with different temperatures (as described
633
above for fruits), and the young fully expanded leaves were sampled for analysis of
634
gene expression.
635
To examine the effect of FaSnRK2.6-RNAi and FaSnRK2.6-OE on temperature-
636
modulated gene expression, the fruits were first transfected with the FaSnRK2.6-
637
RNAi or FaSnRK2.6-OE constructs or the empty vector, and 5 days after the
638
transfection, the fruits were detached and treated with different temperatures (i.e.,
639
40°C for 8 h, or 4°C for 24 h), as described above.
640
Determination of fruit firmness, pigments, soluble sugar content, titratable
22
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
641
acidity, and aroma components
642
Pigment, anthocyanin, total phenol, and flavonoid content of fruits were determined
643
as described (Fuleki et al., 1968; Lees et al.,1971). The soluble sugar content was
644
determined as described (Jia et al., 2011). The total titratable acidity was determined
645
using the acid-base titration method (Kafkas et al., 2007). Titratable acidity was
646
calculated as malic acid. The firmness of fruits transfected by agroinfiltration was
647
determined before freezing. Flesh firmness was measured after removal of fruit skin
648
on opposite sides of each fruit using a GY-4 fruit hardness tester
649
Instrument, Co. Ltp, China). Aroma production by fruits was characterized by SPME
650
(head space solid-phase microextraction) and GC-MS as described (Dong et al., 2013).
651
The relative content of each aroma component was calculated using the peak areas,
652
and expressed as a percentage of the total volatiles.
653
Subcellular localization
654
For subcellular localization of FaSnRK2.6 and FaABI1, the FaSnRK2.6 cDNA
655
sequence
656
CTCGAGATGGATCGGTCTATGCTG-3’
657
GAATTCTAATTGCATACACTATTTCCCCG-3’. The PCR product was then cloned
658
into XhoI/EcoRI-cleaved pEZS-NL transient expression vector (D. Ehrhardt, Carnegie
659
Institution, Stanford, CA). The FaABI1 cDNA sequence was amplified using the
660
forward primer 5’-CTCGAGATGGAGGAGATGTCACC-3’ and the reverse primer
661
5’-GGATCCTCTGTTTTACTTTTAAACTTC-3’, and the PCR product was then
662
cloned into the XhoI/BamHI-cleaved pEZS-NL transient expression vector. Maize
663
protoplast isolation and transformation were carried out according to a protocol
664
described by Sheen et al. (1995), with minor modifications (Li et al., 2012). After
665
pEZS-NL-Fasnrk2.6 or pEZS-NL-Faabi1 were transformed into maize protoplasts,
666
the protoplasts were incubated for 16 h, and then stained with 0.1 mg/mL 4’,6-
667
diamino-phenylindole (DAPI; Sigma-Aldrich) in phosphate-buffered saline for 10
668
min. FaSnRK2.6 and FaABI1 localization patterns were assessed by visualizing GFP
was
amplified
using
the
and
forward
the
（Zhejiang TOP
primer
reverse
primer
5’5’-
23
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
669
fluorescence using a Nikon D-ECLIPSE C1 spectral confocal laser-scanning system,
670
and the nuclei were visualized by DAPI fluorescence. GFP and DAPI were excited at
671
488 nm and 408 nm, respectively. Fluorescence analysis was performed using a
672
Nikon ECLIPSE TE2000-E inverted fluorescence microscope.
673
674
Yeast two-hybrid assays
675
Yeast two-hybrid assays were performed using the Matchmaker GAL4-based Two-
676
hybrid System 3 (Clontech), according to the manufacturer’s instructions. The full-
677
length cDNA sequences of FaABI1 and FaSnRK2.6 were subcloned into the pGADT7
678
and pGBKT7 vector, respectively. All constructs were transformed into yeast strain
679
AH109 by the lithium acetate method, and yeast cells were grown on a minimal
680
medium/-Leu-Trp
681
Transformed colonies were plated onto a minimal medium/-Leu/-Trp/-His/-Ade
682
containing 20 µg/ml 5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside to test for
683
possible interactions. The primers used for yeast two-hybrid assays are listed in
684
Supplemental Table 4.
685
BiFC assays
686
To generate the BiFC constructs, the full-length cDNA sequences of FaABi1 and
687
FaSnRK2.6 were subcloned into the pCambia1300-YFPN and pCambia1300-YFPC
688
vectors. Coexpression was conducted in tobacco leaves as described by K. Schütze
689
(2009). The chimeric fluorescence of the expressed fusion proteins was detected 2-4 d
690
after infiltration. Fluorescence images were acquired using a Nikon D-ECLIPSE C1
691
spectral confocal laser-scanning system. YFP and bright field were excited at 488 nm
692
and 543 nm, respectively. Fluorescence analysis was performed using a Nikon
693
ECLIPSE TE2000-E inverted fluorescence microscope. The primers used for BiFC
694
assays are listed in Supplemental Table 5.
695
Isolation and activity analysis of FaSnRK2.6 promoters
according
to
the
manufacturer’s
instructions
(Clontech).
24
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
696
The promoter sequences of FaSnRK2.6 in fruit and leaf were isolated according to
697
information provided at https://strawberry.plantandfood.co.nz/. The PCR primers were
698
5’-TTAAGCTAATCAGTTTGTTGG-3’
699
TATGAT-3’ and the products were ligated into the pMD19-T simple vector and
700
subsequently transformed into Escherichia coli DH5 . Positive colonies were selected
701
and amplified, and then sequenced by Invitrogen. The fruit and leaf shared the same
702
FaSnRK2.6 promoters. The proFaSnRK2.6 sequence was submitted into the NCBI
703
database (http://www.ncbi.nlm.nih.gov/genbank/), and assigned GenBank accession
704
number
705
(http://www.dna.affrc.go.jp/PLACE/signalscan.html) (Higo K et al., 1999; Prestridge
706
D S et al., 1991).
and
5’-CTCAGAAATGGTGGCTCC-
α
KJ959615.
Promoter
analysis
was
performed
using
PLACE
707
Transient expression assays were employed to test the activity of the promoter. The
708
full-length FaSnRK2.6 promoter was obtained by reverse transcription-polymerase
709
chain
710
AAGCTTTTAAGCTAATCAGTTTGTTGG-3’ (with a HindIII restriction site);
711
reverse, 5’-GGATCCCTCAGAAATGGTGGCTCCTATGAT-3’ (with a
712
restriction site). The cDNA was cloned into the HindIII and BamHI restriction sites of
713
the plant expression vector pBI121, generating the proFaSnRK2.6-GUS construct.
714
The constructs were introduced into Agrobacterium tumefaciens strain AGL0 and the
715
Agrobacterium was infiltrated into fruits (18 DPA) (Jia et al., 2013). After 6 days, the
716
fruits were treated with GUS solution (Gallagher, 1992).
717
Methylation analysis of proFaSnRK2.6
718
BSP-PCR analysis was performed as described (Telias et al., 2011). Briefly, 500 ng of
719
genomic DNA from fruit (18 DPA), leaf, fruit treated for 8 h at 40°C, leaf treated for 8
720
h at 40°C, fruit treated for 24 h at 4°C, and leaf treated for 24 h at 4°C was treated
721
using the EZ DNA Methylation-Gold Kit (Zymo Research). Using the treated DNA as
722
template, eight proFaSnRK2.6 fragments of each sample were amplified using Q5
723
High-fidelity DNA polymerase (NEB), ligated into the PMD19-T simple vector
reaction
using
the
following
primers:
forward,
5’-
BamHI
25
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
724
(TaKaRa), and then sequenced. Sequences of 12 independent clones of each fragment
725
were analyzed using Kismeth (http://katahdin.mssm.edu/kismeth/revpage.pl), and the
726
methylation level of each fragment was calculated. Three biological replicates were
727
performed. Primer sequences are shown in Supporting Information Table 6.
728
Supplemental Materials.
729
The following materials are available in the online version of this article.
730
Supplemental Table 1. Primers used to amplify FaSnRK2.1~FaSnRK2.9 CDSs and
731
GenBank accession numbers.
732
Supplemental Table 2. qPCR primers used in the temporospatial analysis of
733
FaSnRK2.1~FaSnRK2.9.
734
Supplemental Table 3. qPCR primers used to analyze the expression of ripening-
735
related marker genes in FaSnRK2.6 OE and RNAi fruits.
736
Supplemental Table 4. The primers used to amplify FaSnRK2.6 and FaABI1 in yeast
737
two-hybrid assays.
738
Supplemental Table 5. Primers used for FaSnRK2.6 and FaABI1 in BiFC assays.
739
Supplemental Table 6. Primers used to amplify proFaSnRK2.6 in the BSP-PCR
740
analysis.
741
Supplemental Table 7. Bioinformation analysis of sites subjected to proFaSnRK2.6
742
DNA methylation.
743
744
745
ACKNOWLEDGMENTS
746
This work was supported by grants from the National Natural Science Foundation
747
(Grants No. 31101527, 31171921 and 31471851), the Research Fund for the Doctoral
748
Program of Higher Education (Grant No. 20110008120024), and the National High-
749
Tech R&D Program of China (Grant No. 2011AA100204).
750
751
26
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
752
REFERENCES
753
Anantharaman V, Iyer LM, Aravind L (2007) Comparative Genomics of Protists:
754
New Insights into the Evolution of Eukaryotic Signal Transduction and Gene
755
Regulation. Annu Rev Microbiol 61: 453–475
756
Archbold DD (1999) Carbohydrate availability modifies sorbitol dehydrogenase
757
activity of apple fruit. Physiologia Plantarum 105: 391–395
758
Assmann SM (2003) OPEN STOMATA1 opens the door to ABA signaling in
759
Arabidopsis guard cells. Trends in plant science 8: 151–153
760
Bekaert M, Rousset JP (2005) An extended signal involved in eukaryotic− 1
761
frameshifting operates through modification of the E site tRNA. Molecular cell 17:
762
61–68
763
Berüter J, Feusi MES (1995) Comparison of sorbitol transport in excised tissue discs
764
and cortex tissue of intact apple fruit. Journal of plant physiology 146: 95–102
765
Boudsocq M, Barbier-Brygoo H, Laurière C (2004) Identification of nine sucrose
766
nonfermenting 1–related protein kinases 2 activated by hyperosmotic and saline
767
stresses in Arabidopsis thaliana. Journal of Biological Chemistry 279: 41758–41766
768
Bourret RB, Borkovich KA, Simon MI (1991) Signal transduction pathways
769
involving protein phosphorylation in prokaryotes. Annual review of biochemistry 60:
770
401–441
771
Brady CJ (1987) Fruit ripening. Annual review of plant physiology 38: 155–178
772
Bucholc M, Ciesielski A, Goch G, Anielska-Mazur A, Kulik A, Krzywińska E,
773
Dobrowolska G (2011) SNF1–related protein kinases 2 are negatively regulated by a
774
plant–specific calcium sensor. Journal of biological chemistry 286: 3429–3441
775
Capra EJ, Laub MT (2012) Evolution of two–component signal transduction
776
systems. Annual review of microbiology 66: 325–347
777
Chai YM, Jia HF, Li CL, Dong QH, Shen YY (2011) FaPYR1 is involved in
778
strawberry fruit ripening. Journal of Experimental Botany 62: 5079–5089
779
Coombe BG (1976) The development of fleshy fruits. Annual Review of Plant
780
Physiology 27: 207–228
27
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
781
Dong J, Zhang YT, Tang XW, J WM, Han ZH (2013) Differences in volatile ester
782
composition between Fragaria x ananassa and F. vesca and implications for strawberry
783
aroma patterns. Scientia horticulturae 150: 47-53
784
Finnegan EJ, Genger RK, Peacock WJ, Dennis ES (1998) DNA METHYLATION
785
IN PLANTS. Annual Review of Plant Physiology and Plant Molecular Biology 49:
786
223–247
787
Fischer RL, Bennett AB (1991) Role of cell wall hydrolases in fruit ripening. Annual
788
review of plant biology 42: 675–703
789
Folta KM, Davis TM (2006) Strawberry genes and genomics. Critical Reviews in
790
Plant Sciences 25: 399–415
791
Freeman M, Gurdon JB (2002) Regulatory principles of developmental signaling.
792
Annual review of cell and developmental biology 18: 515–539
793
Fujii H, Verslues PE, Zhu JK (2007) Identification of two protein kinases required
794
for abscisic acid regulation of seed germination, root growth, and gene expression in
795
Arabidopsis. The Plant Cell Online 19: 485–494
796
Fujita Y, Nakashima K, Yoshida T, Katagiri T, Kidokoro S, Kanamori N,
797
Yamaguchi-Shinozaki K (2009) Three SnRK2 protein kinases are the main positive
798
regulators of abscisic acid signaling in response to water stress in Arabidopsis. Plant
799
and Cell Physiology 50: 2123–2132
800
Fuleki T, Francis FJ (1968) Quantitative Methods for Anthocyanins Journal of food
801
Science 33: 266–274
802
Gagné S, Cluzet S, Mérillon JM, Gény L (2011) ABA initiates anthocyanin
803
production in grape cell cultures. Journal of plant growth regulation 30: 1–10
804
Gallagher SR (1992) GUS protocols: using the GUS gene as a reporter of gene
805
expression. Academic Press
806
Giovannoni J (2001) Molecular biology of fruit maturation and ripening. Annual
807
review of plant biology 52: 725–749
808
Giovannoni J (2004) Genetic regulation of fruit development and ripening. The Plant
809
Cell Online 16: 170–180
28
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
810
Giribaldi M, Gény L, Delrot S, Schubert A (2010) Proteomic analysis of the effects
811
of ABA treatments on ripening Vitis vinifera berries. Journal of experimental botany
812
61: 2447–2458
813
Hadi MAME, Zhang FJ, Wu FF, Zhou CH, Tao J (2013) Advances in fruit
814
aroma volatile research. Molecules 18: 8200-8229
815
Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis–acting regulatory
816
DNA elements (PLACE) database:1999. Nucleic Acids Research 27: 297–300
817
Hirayama T, Shinozaki K (2010) Research on plant abiotic stress responses in the
818
post genome era: past, present and future. The Plant Journal 61: 1041–1052
819
Hrabak EM, Chan CW, Gribskov M, Harper JF, Choi JH, Halford N, Harmon
820
AC (2003) The Arabidopsis CDPK–SnRK superfamily of protein kinases. Plant
821
physiology 132: 666–680
822
Imes D, Mumm P, Böhm J, Al-Rasheid KA, Marten I, Geiger D, Hedrich R
823
(2013) Open stomata 1 (OST1) kinase controls R–type anion channel QUAC1 in
824
Arabidopsis guard cells. The Plant Journal 74: 372–382
825
Jia HF, Chai YM, Li CL, Lu D, Luo JJ, Qin L, Shen YY (2011) Abscisic acid plays
826
an important role in the regulation of strawberry fruit ripening. Plant Physiology 157:
827
188–199
828
Jia HF, Lu D, Sun JH, Li CL, Xing Y, Qin L, Shen YY (2013) Type 2C protein
829
phosphatase ABI1 is a negative regulator of strawberry fruit ripening. Journal of
830
experimental botany 64: 1677–1687
831
Jia HF, Wang YH, Sun MZ, Li BB, Han Y, Zhao YX, Jia WS (2013) Sucrose
832
functions as a signal involved in the regulation of strawberry fruit development and
833
ripening. New Phytologist 198: 453–465
834
Jiang M, Zhang J (2001) Effect of abscisic acid on active oxygen species,
835
antioxidative defence system and oxidative damage in leaves of maize seedlings.
836
Plant and Cell Physiology 42: 1265–1273
‐
29
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
837
Julian IS, Gethyn JA, Veronique H, June MK, David W (2001) GUARD CELL
838
SIGNAL TRANSDUCTION. Annual Review of Plant Physiology and Plant
839
Molecular Biology 52: 627–658
840
Kafkas E, Koşar M, Paydaş S, Kafkas S, Başer KHC (2007) Quality characteristics
841
of strawberry genotypes at different maturation stages. Food Chemistry 100: 1229–
842
1236
843
Kim TH, Maik BĆ (2010) Guard cell signal transduction network: advances in
844
understanding abscisic acid, CO2, and Ca2+ signaling. Annual review of plant biology
845
61: 561
846
Klee HJ, Giovannoni JJ (2011) Genetics and control of tomato fruit ripening and
847
quality attributes. Annual review of genetics 45: 41–59
848
Kobayashi Y, Murata M, Minami H, Yamamoto S, Kagaya Y, Hobo T, Hattori T
849
(2005) Abscisic acid activated SNRK2 protein kinases function in the gene regulation
850
pathway of ABA signal transduction by phosphorylating ABA response element
851
binding factors. The Plant Journal 44: 939–949
852
Koyama K, Sadamatsu K, Goto-Yamamoto N (2010) Abscisic acid stimulated
853
ripening and gene expression in berry skins of the Cabernet Sauvignon grape.
854
Functional & integrative genomics 10: 367–381
855
Kulik A, Wawer I, Krzywińska E, Bucholc M, Dobrowolska G (2011) SnRK2
856
protein kinases—key regulators of plant response to abiotic stresses. Omics: a journal
857
of integrative biology 15: 859–872
858
Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation–competent
859
Arabidopsis genomic library in Agrobacterium. Nature Biotechnology 9: 963–967
860
Lees DH, Francis FJ (1971) Quantitative methods for anthocyanins. Journal of Food
861
Science 36: 1056–1060
862
Leung J, Giraudat J (1998) Abscisic acid signal transduction. Annual review of
863
plant biology 49: 199–222
‐
‐
‐
30
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
864
Li B, Zhao Y, Liang L, Ren H, Xing Y, Chen L, Jia W (2012) Purification and
865
characterization of ZmRIP1, a novel reductant–inhibited protein tyrosine phosphatase
866
from maize. Plant physiology 159: 671–681
867
Llop-Tous I, Domínguez-Puigjaner E, Vendrell M (2002) Characterization of a
868
strawberry cDNA clone homologous to calcium dependent protein kinases that is
869
expressed during fruit ripening and affected by low temperature. Journal of
870
experimental botany 53: 2283–2285
871
Lu SX, Hrabak EM (2013) The myristoylated amino–terminus of an Arabidopsis
872
calcium–dependent protein kinase mediates plasma membrane localization. Plant
873
molecular biology 82: 267–278
874
Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E (2009)
875
Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science
876
324: 1064–1068
877
Mao X, Zhang H, Tian S, Chang X, Jing R (2010) TaSnRK2 4, an SNF1–type
878
serine/threonine protein kinase of wheat (Triticum aestivum L), confers enhanced
879
multistress tolerance in Arabidopsis. Journal of experimental botany 61: 683–696
880
Mustilli AC, Merlot S, Vavasseur A, Fenzi F, Giraudat J (2002) Arabidopsis
881
OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and
882
acts upstream of reactive oxygen species production. The Plant Cell Online 14: 3089–
883
3099
884
Nishimura N, Sarkeshik A, Nito K, Park SY, Wang A, Carvalho PC, Schroeder JI
885
(2010) PYR/PYL/RCAR family members are major in
886
phosphatase 2C interacting proteins in Arabidopsis. The Plant Journal 61: 290–299
887
Nitsch JP (1953) The physiology of fruit growth. Annual Review of Plant Physiology
888
4: 199–236
889
Owen SJ, Lafond MD, Bowen P, Bogdanoff C, Usher K, Abrams SR (2009)
890
Profiles of abscisic acid and its catabolites in developing Merlot grape (Vitis vinifera)
891
berries. American Journal of Enology and Viticulture 60: 277–284
892
Park SY, Fung P, Nishimura N, Jensen DR, Fujii H, Zhao Y, Cutler SR (2009)
‐
‐
‐ vivo
ABI1 protein
31
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
893
Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of
894
START proteins. Science 324: 1068–1071
895
Park YS, Hong SW, Oh SA, Kwak JM, Lee HH, Nam HG (1993) Two putative
896
protein kinases from Arabidopsis thaliana contain highly acidic domains. Plant
897
molecular biology 22: 615–624
898
Prestridge DS (1991) SIGNAL SCAN: A computer program that scans DNA
899
sequences for eukaryotic transcriptional elements. CABIOS 7: 203–206
900
Santiago J, Dupeux F, Betz K, Antoni R, Gonzalez-Guzman M, Rodriguez L,
901
Rodriguez PL (2012) Structural insights into PYR/PYL/RCAR ABA receptors and
902
PP2Cs. Plant Science 182: 3–11
903
Schmittgen TD, Livak KJ (2008) Analyzing real–time PCR data by the comparative
904
CT method. Nature protocols 3: 1101–1108
905
Schroeder JI, Allen GJ, Hugouvieux V, Kwak JM, Waner D (2001) Guard cell
906
signal transduction. Annual review of plant biology 52: 627–658
907
Schütze K, Harter K, Chaban C (2009) Bimolecular fluorescence complementation
908
(BiFC) to study protein–protein interactions in living plant cells. In Plant Signal
909
Transduction 479: 189–202
910
Seymour GB, Østergaard L, Chapman NH, Knapp S, Martin C (2013) Fruit
911
development and ripening. Annual review of plant biology 64: 219–241
912
Seymour GB, Taylor JE, Tucker GA (1993) Biochemistry of fruit ripening.
913
Chapman & Hall
914
Sheen J, Hwang S, Niwa Y, Kobayashi H, Galbraith DW (1995) Green fluorescent
915
protein as a new vital marker in plant cells. The Plant Journal 8: 777–784
916
Shukla V, Mattoo AK (2008) Sucrose non–fermenting 1–related protein kinase 2
917
(SnRK2): a family of protein kinases involved in hyperosmotic stress signaling.
918
Physiology and molecular biology of plants 14: 91–100
919
Shulaev V, Cortes D, Miller G, Mittler R (2008) Metabolomics for plant stress
920
response. Physiologia Plantarum 132: 199–208
‐
32
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
921
Sirichandra C, Davanture M, Turk BE, Zivy M, Valot B, Leung J, Merlot S
922
(2010) The Arabidopsis ABA–activated kinase OST1 phosphorylates the bZIP
923
transcription factor ABF3 and creates a 14–3–3 binding site involved in its turnover.
924
PLoS One 5: e13935
925
Soon FF, Ng LM, Zhou XE, West GM, Kovach A, Tan ME, Xu HE (2012)
926
Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C
927
phosphatases. Science 335: 85–88
928
Sun L, Sun Y, Zhang M, Wang L, Ren J, Cui M, Leng P (2012) Suppression of 9–
929
cis–epoxycarotenoid dioxygenase, which encodes a key enzyme in abscisic acid
930
biosynthesis, alters fruit texture in transgenic tomato. Plant physiology 158: 283–298
931
Telias A, Lin-Wang K, Stevenson DE, Cooney JM, Hellens RP, Allan AC, Hoovr
932
EE, Bradeen JM ( 2011) Apple skin patterning is associated with differential
933
expression of MYB10. BMC Plant Biol 11: 93–107
934
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the
935
sensitivity of progressive multiple sequence alignment through sequence weighting,
936
position–specific gap penalties and weight matrix choice. Nucleic acids research 22:
937
4673–4680
938
Ulrich R (1958) Postharvest physiology of fruits. Annual Review of Plant Physiology
939
9: 385–416
940
Umezawa T, Yoshida R, Maruyama K, Yamaguchi-Shinozaki K, Shinozaki K
941
(2004) SRK2C, a SNF1–related protein kinase 2, improves drought tolerance by
942
controlling stress–responsive gene expression in Arabidopsis thaliana. Proceedings of
943
the National Academy of Sciences of the United States of America 101: 17306–17311
944
Vilela B, Moreno-Cortés A, Rabissi A, Leung J, Pagès M, Lumbreras V (2013)
945
The maize OST1 kinase homolog phosphorylates and regulates the maize SNAC1–
946
type transcription factor. PloS one 8: e58105
947
Vlad F, Rubio S, Rodrigues A, Sirichandra C, Belin C, Robert N, Merlot S (2009)
948
Protein phosphatases 2C regulate the activation of the Snf1–related kinase OST1 by
949
abscisic acid in Arabidopsis. The Plant Cell Online 21: 3170–3184
33
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
950
Wang M, Yuan F, Hao H, Zhang Y, Zhao H, Guo A, Xie CG (2013) BolOST1, an
951
ortholog of Open Stomata 1 with alternative splicing products in Brassica oleracea,
952
positively modulates drought responses in plants. Biochemical and biophysical
953
research communications 442: 214–220
954
Wang P, Xue L, Batelli G, Lee S, Hou YJ, Van-Oosten MJ, Zhu JK (2013)
955
Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and
956
reveals the effectors of abscisic acid action. Proceedings of the National Academy of
957
Sciences 110: 11205–11210
958
White MA, Anderson RG (2005) Signaling networks in living cells. Annu Rev
959
Pharmacol Toxicol 45: 587–603
960
Xue S, Hu H, Ries A, Merilo E, Kollist H, Schroeder JI (2011) Central functions of
961
bicarbonate in S type anion channel activation and OST1 protein kinase in CO2 signal
962
transduction in guard cell. The EMBO journal 30: 1645–1658
963
Yang T, Peng H, Whitaker BD, Conway WS (2012) Characterization of a
964
calcium/calmodulin–regulated SR/CAMTA gene family during tomato fruit
965
development and ripening. BMC plant biology 12: 19
966
Yoshida R, Hobo T, Ichimura K, Mizoguchi T, Takahashi F, Aronso J, Shinozaki
967
K (2002) ABA–activated SnRK2 protein kinase is required for dehydration stress
968
signaling in Arabidopsis. Plant and Cell Physiology 43: 1473–1483
969
Yoshida T, Nishimura N, Kitahata N, Kuromori T, Ito T, Asami T, Hirayama T
970
(2006) ABA–hypersensitive germination3 encodes a protein phosphatase 2C
971
(AtPP2CA) that strongly regulates abscisic acid signaling during germination among
972
Arabidopsis protein phosphatase 2Cs. Plant Physiology 140: 115–126
973
Zheng Z, Xu X, Crosley RA, Greenwalt SA, Sun Y, Blakeslee B, Greene TW
974
(2010) The protein kinase SnRK2. 6 mediates the regulation of sucrose metabolism
975
and plant growth in Arabidopsis. Plant Physiology 153: 99–113
976
977
Karlova R, Chapman N, David K, Angenent GC, Seymour GB and DE Maagd
RA (2014) Transcriptional control of fleshy fruit development Journal of
Experimental Botany 65: 4527-454
978
‐
34
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H
Genotypes
Parameters
Firmness (Kg/cm2)
H
H
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Anthocyanin
content
(mg/g.FW)
Flavonoid content (μg/g.FW)
Total
phenol
content
(μg/g.FW)
Fructose content (mg/g.FW)
Sucrose content (mg/g.FW)
Glucose content (mg/g.FW)
Total titratable acid content
(%)
Hexanal
2-Hexenal
Octanal
1-Hexanol
1-Pentanol, 2-methyl
1,5-Pentanediol
2-Pentanol
1-Pentanol, 2-ethyl-4-methyl
1,3-Propanediol, 2-ethyl-2(hydroxymethyl)
4H-Pyran-4-one, 2,3-
Control-RNAi
OE
Control-RNAi
RNAi
2.815±0.262
6.990±0.126**
6.185±0.117
2.155±0.158**
0.110±0.005
0.039±0.002**
0.040±0.003
0.096±0.004**
2.073±0.057
3.795±0.091
3.142±0.160**
4.661±0.081**
4.346±0.095
6.367±0.090
2.790±0.080**
4.463±0.064**
15.825±1.163
26.328±1.225
17.921±1.137
1.127±0.036
16.105±1.297
25.659±1.015
18.021±0.129
1.125±0.089
13.635±1.277
21.865±1.365
16.011±1.021
1.401±0.021
14.348±0.364
21.118±0.965
15.213±0.361
1.263±0.062
0.437±0.002
1.106±0.042
0.000±0.000
0.422±0.016
0.000±0.000
4.662±0.142
0.000±0.000
0.000±0.000
5.520±0.091
0.743±0.005**
2.585±0.008**
1.520±0.001**
0.731±0.001**
2.200±0.018**
5.881±0..029**
2.230±0.019**
0.720±0.009**
0.000±0.000**
1.510±0.062
4.833±0.009
0.000±0.000
1.550±0.003
3.227±0.004
6.476±0.015
0.000±0.000
0.000±0.000
0.000±0.000
0.653±0.011**
1.800±0.008**
0.000±0.000
0.717±0.023**
0.000±0.000**
5.420±0.109**
0.000±0.000
0.000±0.000
7.050±0.017**
8.438±0.089
1.600±0.041**
5.656±0.593
9.672±0.099**
Notes
Cell wall metabolismrelated parameter
Pigment
metabolismrelated compounds
Sugar metabolism-realted
compounds
Acid metabolism-realted
parameter
Aroma metabolism-related
compounds.
Data
are
expressed as percentage of
the total volatiles (%).
35
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
979
dihydro-3,5-dihydroxy-6methyl
2-Furancarboxaldehyde, 5(hydroxymethyl)
2,4(1H,3H)Pyrimidinedione, 5-methyl
Hexanoic acid, methyl ester
Acetic acid, hexyl ester
Butanoic acid, 3-oxo-, propyl
ester
2-Hexen-1-ol
Diethyl Phthalate
1,2-Benzenedicarboxylic
acid, butyl octyl ester
1,2-Benzenedicarboxylic
acid, bis(2-methylpropyl)
ester
12.416±0.123
2.350±0.090**
6.677±0.311
15.542±0.821**
1.230±0.030
1.370±0.005
2.140±0.048
2.160±0.031
1.030±0.006
0.653±0.001
1.800±0.068
0.000±0.000**
0.000±0.000**
0.000±0.000**
0.000±0.000
0.000±0.000
0.000±0.000
1.020±0.005**
2.530±0.050**
2.360±0.013**
3.692±0.017
1.857±0.009
3.337±0.027
1.850±0.099**
0.680±0.002**
3.419±0.015
1.542±0.041
1.450±0.011
2.928±0.009
3.253±0.007**
2.071±0.016**
2.777±0.039
4.073±0.092
2.363±0.024**
1.415±0.004
2.219±0.011**
Table 1 Effect of FaSnRK2.6-OE and FaSnRK2.6-RNAi on the major fruit ripening-related parameters
980
The OE and RNAi constructs were transfected into fruits at 18 DPAdays post anthesis, and 7 (for RNAi) or 12 (for OE)
981
days after the transfection, fruits were detached and used for the analysis. Values are means +SD of four samples (each
982
sample includes 5 fruits). ** denote significant differences compared with the control sample (i.e. OE-C or RNAi-C) at P <
983
0.01, respectively, using Student’s t-test
36
984
FIGURE LEGENDS
985
Table 1 Effect of FaSnRK2.6-OE and FaSnRK2.6-RNAi on major fruit ripening-
986
related parameters
987
The OE and RNAi constructs were transfected into fruits at 18 DPA, and 7 (for RNAi)
988
or 12 (for OE) days after the transfection, fruits were detached and used for the
989
analysis. Values are means +SD of four samples (each sample includes 5 fruits). **
990
denotes significant differences compared with the control sample (i.e. OE-C or RNAi-
991
C) at P < 0.01, respectively, using Student’s t-test.
992
993
Figure 1. Phylogenetic analysis of SnRK2.6 proteins.
994
(A) Sequence alignments of the deduced amino acid sequences of FaSnRK2s. The
995
deduced amino acid sequences of FaSnRK2.1-FaSnRK2.9 were aligned using Clustal
996
X 2.0.12 software with default settings. The alignments were edited and marked using
997
GeneDoc. Black and light gray shading indicate identical and similar amino acid
998
residues, respectively. The predicted functional domains are boxed. (B) Phylogenic
999
analysis of SnRK2 homologs from various plant species. The phylogenetic trees were
1000
constructed using the neighbor-joining (N-J) method in MEGA6 software with 1000
1001
bootstrap replicates. The numbers at nodes represent bootstrap value. OST1 and its
1002
ortholog in strawberry are marked with black dots. Abbreviation of species names: At,
1003
Arabidopsis thaliana; Zm, Zea mays; Tp, Triticum polonicum L; St, Solanum
1004
tuberosum; Ta, Triticum aestivum; Np, Nicotiana plumbaginifolia.
1005
Figure 2. Temporospatial pattern of FaSnRK2 expression in strawberry fruits.
1006
(A) qRT-PCR analysis of FaSnRK2.6 expression in different organs of the strawberry
1007
plant. (B) Phenotypes showing different developmental stages of strawberry fruit. SG,
1008
small green fruit; MG, middle green fruit; LG, large green fruit; W, white fruit; IR,
1009
initial reddening; and FR, full reddening. (C) qPCR analysis of FaSnRK2 expression
1010
in different developmental stages of strawberry fruit. Labels below bars denote the
37
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1011
corresponding developmental stages as explained in (B). For (A) and (C), qRT-PCR
1012
was conducted using FaACTIN as an internal control. Values are means + SD of five
1013
biolgical replicates.
1014
Figure 3. qRT-PCR analysis of FaSnRK2.6 expression in response to ABA and
1015
sucrose treatments in fruits and leaves.
1016
(A) FaSnRK2.6 expression in response to ABA in fruits. Detached fruits in the large
1017
green fruit substage (i.e., LG, corresponding to 18 DPA) were treated with various
1018
concentrations of ABA for 1 or 6 h, and then FaSnRK2.6 expression was examined.
1019
(B) FaSnRK2.6 expression in response to ABA treatment in leaves. Young, fully
1020
expanded leaves were treated with various concentrations of ABA for 1 or 6 h, and
1021
then the FaSnRK2.6 expression level was examined. (C) FaSnRK2.6 expression in
1022
response to sucrose treatment in fruits. Detached fruits in the LG substage were
1023
treated with various concentrations of sucrose for 1 or 6 h, and then the FaSnRK2.6
1024
expression level was examined. qRT-PCR was conducted using FaACTIN as an
1025
internal control. Values are means + SD of four bilogical replicates. * and **
1026
respectively denote significant differences compared with the control sample (i.e., the
1027
0 concentration) at P < 0.05 and P < 0.01, according to Student’s t-test.
1028
Figure 4. The effect of FaSnRK2.6 over-expression (OE) and interference (RNAi) on
1029
the levels of FaSnRK2.6 transcript and strawberry fruit development and ripening.
1030
(A)
1031
transcript. FaSnRK2.6-OE and FaSnRK2.6-RNAi were performed as described in
1032
Materials and Methods. The OE and RNAi constructs were injected into the fruits at
1033
18 DPA. Levels of FaSnRK2.6 were examined at 30 DPA for the OE samples and at
1034
25 DPA for the RNAi samples. Control samples were transfected with the empty
1035
vector (pCambia1304). qRT-PCR was conducted using FaACTIN as an internal
1036
control. Values are means + SD of 4 biological replicates. ** denotes significant
1037
difference at P < 0.01 using Student’s t-test. Effect of FaSnRK2.6-OE on strawberry
1038
fruit development and ripening.
Effect of FaSnRK2.6-OE and FaSnRK2.6-RNAi on the levels of FaSnRK2.6
38
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1039 (B)
Upper panel, phenotype of fruits immediately after construct injection. Note
1040
the color change caused by the injection. Lower panel, note that when the control fruit
1041
became red, the fruit of OE was still in the W stage. (C) Effect of FaSnRK2.6-RNAi on
1042
strawberry fruit development and ripening. Note that when the control fruit was at the
1043
W stage, the fruit of RNAi had already become red.
1044
1045
Figure 5. The effect of FaSnRK2.6 over-expression (OE) and RNA interference
1046
(RNAi) on the expression of ripening-related genes.
1047
FaSnRK2.6-OE and FaSnRK2.6-RNAi fruits were generated as described in Materials
1048
and Methods. The OE and RNAi constructs were injected into fruits at 18 DPA, and
1049
gene expression was examined at 30 and 25 DPA, respectively, using qRT-PCR, with
1050
FaACTIN as the internal control. OE-C and RNAi-C denote controls for the OE or
1051
RNAi fruit (i.e., fruit transformed with the corresponding empty vector). Fa, Fragaria
1052
x ananassa; C4H, cinnamic acid 4-hydroxylase; 4CL1 and 4CL2, 4-coumaroyl-CoA
1053
ligase 1 and 4-coumaroyl-CoA ligase 2; CHS, chalcone synthase; CHI, chalcone
1054
isomerase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonolreductase; UFGT,
1055
UDP-glycose flavonoid 3-O-glycosyltransferase; QR, quinineoxidoreductase; OMT,
1056
O-methyltransferase; PE, pectinesterase; PL, pectatelyase; PG, polygalacturonase;
1057
CEL, cellulose; GAL1 and GAL2, beta-galactosidase 1 and beta-galactosidase 2; XYL1,
1058
beta-xylosidase1; and EXP1,2,3,4,5, expansin1,2,3,4,5. Values are means +SD of four
1059
biological replicates. * and ** denote significant differences compared with the
1060
control sample (i.e., OE-C or RNAi-C) at P < 0.05 and P < 0.01, respectively, using
1061
Student’s t-test.
1062
Figure 6. Subcellular localization of FaSnRK2.6 and FaABI1 and physical interaction
1063
between FaSnRK2.6 and FaABI1.
1064
(A) The subcellular localization of FaSnRK2.6 and FaABI1. pEZS-NL-Fasnrk2.6 or
1065
pEZS-NL-Faabi1 constructs were transdformed into maize protoplasts and
39
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1066
fluorescence was observed by confocal microscopy as described in Materials and
1067
Methods. Bars = 8 µm.
1068
(B) Yeast two-hybrid analysis of the physical interaction between FaSnRK2.6 and
1069
FaABI1. The protein interaction was examined using various combinations of prey
1070
and bait vectors. All tests were conducted on media containing adenine ‘+Ade’
1071
(/+Leu/+Trp/-His/-Ade)
1072
Interactions were determined based on cell growth and were confirmed by a α-gal
1073
assay on medium lacking adenine (/-Leu/-Trp/-His/-Ade). Dilutions (1, 10-1, and 10-2)
1074
of saturated cultures were spotted onto the plates.
1075
(C) BiFC analysis of the physical interaction between FaSnRK2.6 and FaABI1.
1076
FaSnRK2.6 and FaABI1 were fused with either the C or N terminus of YFP
1077
(respectively designated as YFPc or YFPN). Different combinations of the fused
1078
constructs were co-transformed into tobacco cells and the cells were then visualized
1079
using confocal microscopy as described in Materials and Methods. YFP and bright
1080
field were excited at 488 nm and 543 nm, respectively. Bars =20 μm.
1081
Figure 7. Effect of temperature on strawberry fruit development and ripening.
1082
(A) Photographs showing the progression of strawberry fruit development and
1083
ripening under two different temperatures. Strawberry plants were first grown under
1084
normal temperature conditions (i.e., 25°C/18°C, day/night) until fruit setting and were
1085
then subjected to one of two different temperature conditions, i.e., 32°C/20°C,
1086
day/night or 20°C/12°C, day/night, with other conditions left unchanged. Successive
1087
photographs show the developmental progression of a representative fruit, with the
1088
numbers indicating the duration (in days) of treatment. (B) The number of fully
1089
ripened fruits was counted for a given number of plants subjected either to high
1090
(32°C/20°C) or low (20°C/12°C) temperature treatments. The horizontal distance
1091
between the two lines represents differences in the number of days to ripening under
1092
the two different temperature conditions.
1093
Figure 8. FaSnRK2.6 expression in response to temperature treatments in fruits and
or
lacking
adenine
‘-Ade’
(/-Leu/-Trp/-His/-Ade).
40
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1094
leaves.
1095
In (A) and (B), fruits were detached at the LG substage (corresponding to 18 DPA)
1096
and then treated with high (40°C) or low (4°C) temperature for the indicated period of
1097
time. Samples were then subjected to qRT-PCR analysis to evaluate FaSnRK2.6
1098
expression. For high temperature treatment (A), the fruit samples were incubated at
1099
40°C for 0 to 8 h, and for low temperature treatment (B), the fruit samples were
1100
treated at 4°C for 0 to 48 h. (C) High temperature treatment in leaves. (D) Low
1101
temperature treatment in leaves. For (C) and (D), young, fully expanded leaves were
1102
used and the method for temperature treatment was the same as described in (A) and
1103
(B). qRT-PCR was conducted using FaACTIN as an internal control. Values are means
1104
+ SD of 3 biological replicates. * and ** respectively denote significant differences at
1105
P < 0.05 and P < 0.01 using Student’s t-test, as compared with the control sample (i.e.,
1106
0 h, 25°C).
1107
Figure 9. The effect of FaSnRK2.6 over-expression (OE) and RNA interference
1108
(RNAi) on the expression of temperature-responsive genes.
1109
The FaSnRK2.6-OE and FaSnRK2.6-RNAi constructs were generated and
1110
transformed into fruits at 18 DPA, as described in Materials and Methods. Five days
1111
after transformation, the fruits were detached and used to analyze temperature-
1112
modulated gene expression. (A) Effect of FaSnRK2.6-OE and FaSnRK2.6-RNAi on
1113
the level of FaSnRK2.6 transcript in fruits. High temperature treatment: 40°C, 8 h;
1114
low temperature treatment: 4°C, 24 h; Non treatment: gene expression was analyzed
1115
immediately after fruits were detached; EV, empty vector; EV-0 h, OE-0 h, and RNAi
1116
0 h all indicate ‘non treatment’. C, control temperature (i.e., 25°C); T, high or low
1117
temperature treatment. (B) Effects of FaSnRK2.6-OE and FaSnRK2.6-RNAi on
1118
FaSnRK2.6 expression in response to high temperature treatment. C: control
1119
temperature (i.e., 25°C); 40°C: high temperature (40°C) treatment for 8 h. (C) Effects
1120
of FaSnRK2.6-OE and FaSnRK2.6-RNAi on FaSnRK2.6 expression in response to
1121
low temperature treatment. C: control temperature (i.e., 25°C ); 4°C: low (4°C)
1122
treatment for 24 h. CHS, chalcone synthase; CHI, chalcone isomerase; UFGT, UDP41
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1123
glycose
flavonoid 3-O-glycosyltransferase; PE,
1124
xylosidase1; and QR, quinineoxidoreductase. qRT-PCR was conducted using
1125
FaACTIN as an internal control. Values are means + SD of four biological replicates.
1126
Different lowercase letters indicate significant differences according to Fisher’s LSD
1127
(P <0.05).
1128
Figure 10. Cytosine methylation analysis of proFaSnRK2.6 in fruit and leaf.
1129
(A) Confirmation that proFaSnRK2.6 is active. proFaSnRK2.6 was inserted into the
1130
pBI121 vector to drive the GUS reporter gene. The construct was introduced into
1131
fruits (18 DPA), and proFaSnRK2.6 activity was estimated by GUS staining six days
1132
later. As a control, the pBI121 empty vector with the GUS promoter deleted was used.
1133
(B) Cytosine methylation levels of proFaSnRK2.6 in fruits and leaves were estimated
1134
using BSP-PCR. A total of 500 ng of genomic DNA extracted from fruits (18 DPA)
1135
or leaves was used to perform bisulfite modification, and the product was used as a
1136
template to amplify proFaSnRM2.6 using eight primer pairs, and 12 independent
1137
clones
1138
(http://katahdin.mssm.edu/kismeth/revpage.pl). Control: control temperature (25°C);
1139
high temperature treatment，40°C for 8 h; low temperature, 4°C for 24 h. *, site of
1140
difference between fruit and leaf; black triangle, site of difference as caused by
1141
temperature treatment.
1142
Figure 11. Diagram of FaSnRK2.6 signaling in relation to the regulation of
1143
strawberry fruit development and ripening.
1144
Levels of FaSnRK2.6 transcript decrease during the progression of strawberry fruit
1145
development and ripening. Overexpression and RNAi-mediated suppression of
1146
FaSnRK2.6 expression respectively delay and accelerate the onset of strawberry fruit
1147
ripening. ABA and high temperature downregulate the expression of FaSnRK2.6,
1148
thereby promoting strawberry fruit ripening. As seed maturation and dispersal can be
1149
regarded as a strategy for plants to withstand environmental stresses, the regulation of
from
each
PCR
were
sequenced
pectinesterase; XYL1, beta-
and
analyzed
using
Kismeth
42
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1150
strawberry fruit ripening by FaSnRK2.6 embodies a role of FaSnRK2.6 in stress
1151
signaling.
43
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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