Arabidopsis AtSUC2 and AtSUC4, encoding sucrose transporters, are required for abiotic stress tolerance in an ABA-dependent pathway Xue Gonga,b,†, Mingli Liua,†, Lijun Zhanga,†,Yanye Ruana, Rui Dinga, Yuqi Jic, Ning Zhanga, Shaobin Zhanga, John Farmerd and Che Wanga,* a
College of Biological Science and Technology, Shenyang Agricultural University, Shenyang, 100866,
China b
Corn Research Institute, Liaoning Academy of Agricultural Sciences, Shenyang 110161, China
c
College of Agriculture and Biotechnology, China Agricultural University, Beijing 100094, China
d
College of Land and Environment, Shenyang Agricultural University, Shenyang, 100866, China
*Corresponding author, e-mail: [email protected] †
These authors contributed equally to this work.
Sucrose transporters (SUCs or SUTs) play a central role, as they orchestrate sucrose allocation both intracellularly and at the whole plant level. Previously, we found AtSUC4 mutants changing sucrose distribution under drought and salt stresses. Here, we systematically examined the role of Arabidopsis AtSUC2 and AtSUC4 in response to abiotic stress. The results showed that significant induction of AtSUC2 and AtSUC4 in salt, osmotic, low temperature and exogenous ABA treatments by public microarray data and real-time quantitative reverse transcription PCR (qRT-PCR) analyses. The loss-of-function mutation of AtSUC2 and AtSUC4 led to hypersensitive responses to abiotic stress and ABA treatment in seed germination and seedling growth. These mutants also showed higher sucrose content in shoots and lower sucrose content in roots, as compared to that in wild-type plants, and inhibited the ABA-induced expression of many stress-responsive genes and ABA-responsive genes, especially ABFs and ABF-downstream and upstream genes. The loss-of-function mutant of AtSUC3, a unique putative sucrose sensor, reduced the expression of AtSUC2 and AtSUC4 in response to abiotic stresses and ABA. These findings confirmed that AtSUC2 and AtSUC4 are important regulators in plant abiotic stress tolerance by the use of an ABA signaling pathway, which may be crossed with sucrose signaling.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ppl.12225 This article is protected by copyright. All rights reserved
Abbreviations – Abscisic acid, ABA; ABSCISIC ACID-INSENSITIVE2, ABI2; Arabidopsis Biological Resource Center, ABRC; ABA repressor 1, ABR1; High-performance liquid chromatography, HPLC; Impaired sucrose induction, isi; Left genomic primer, LP; Right genomic primer, RP; Real-time quantitative reverse transcription PCR, qRT-PCR; Reverse transcription PCR, RT-PCR; Sucrose, Suc; Sieve elements, SEs; Sugar insensitive, sis; Sucrose non-fermenting 1-related protein kinase, SnRKs; Sucrose phosphate synthase, SPS; Sucrose synthase, SuSy; Sucrose transporters, SUCs or SUTs; Wild type, WT
Introduction Plant growth and productivity are greatly affected by environmental stresses such as high salinity, dehydration, and low temperature. The responses and adaptations of plants to these stresses can occur at the molecular, cellular, physiological, and biochemical levels (Chinnusamy et al. 2005, Urano et al. 2010, Fujii and Zhu 2012). As a metabolite, sucrose plays an important role in stress tolerance, serving as an osmolyte, a signaling molecule and/or a nutritional substance (Yu 1999, Gupta and Kaur 2005, Ruan et al. 2010). The expressions of some sucrose related genes, such as SuSy (sucrose synthase) (Baud et al. 2004), SPS (sucrose phosphate synthase) (Pelleschi et al. 1997) and SnRK2s (sucrose non-fermenting 1-related protein kinase2) (Fujii and Zhu 2012, Zhang et al. 2011) have been reported to change in response to abiotic stresses. Sucrose transporters (SUCs or SUTs) are an important exporter of photosynthetically produced sugar, principally sucrose, from higher plant source leaves to sink tissues. In Arabidopsis, 9 AtSUCs have been identified and researched on some of their expressions and functions. AtSUC2, is expressed in companion cells collection and transport phloem in source leaves (Truernit and Sauer 1995, Martens et al. 2006) and functions in loading sucrose into the phloem sieve elements (SEs), as determined by tissue-specific complementation of different promoters (Srivastava et al. 2008, 2009a) and by
14
C labelling studies (Gottwald et al. 2000). This protein is also found within the vascular
bundles of sink tissues such as stamens, siliques, and roots (Truernit and Sauer 1995, Stadler and Sauer 1996), suggesting a role in the retrieval of sucrose that leaks out of sieve elements during long-distance transport (Stadler and Sauer 1996). When planted without sucrose, Atsuc2 mutant seedlings are smaller than wild-type (WT) seedlings (Gottwald et al. 2000). AtSUC4 and AtSUC3 are also expressed in minor veins of source leaves of mature plants (Weise et al. 2000, Barker et al. 2000, Aoki et al. 2012), suggesting an involvement in phloem loading of sucrose in source organs. The sucrose/proton symporter, AtSUC4, is also expressed in vacuoles and most likely regulates the transport and vacuolar storage of photosynthetically derived sucrose (Endler et al. 2006, Schulz et al. 2011).
This article is protected by copyright. All rights reserved
Sporadic reports have appeared regarding the role of SUTs in plant responses to abiotic stresses. The expression of AgSUT1, which codes for the high affinity of AgSUT1 sucrose/H+ transporter in celery (Apium graveolens), was decreased in all plant organs in response to salinity stress—a more marked decrease in roots may have reflected a decreased metabolic demand for sucrose in response to stress (Noiraud et al. 2000). Among the five sucrose transporters identified in rice (Oryza sativa cv. Nipponbare), only OsSUT2 expression was up-regulated following exposure to drought and salinity treatments (Ibraheem et al. 2011, Lundmark et al. 2006) found a low temperature induced up-regulation of AtSUC1 and AtSUC2 when analyzing the role of the sucrose-phosphate synthase (SPSox) in carbon partitioning of Arabidopsis at low temperature. Recent studies indicate that Aspen (Populus tremula) transformed with PtaSUT4-RNAi wilt when exposed to a short-term drought, indicating a role for PtaSUT4 in drought tolerance (Frost et al. 2012). Although these results indicate that SUCs are involved in plant response abiotic stress, it is not clear that the potential stress tolerance functions of the SUCs that control SUC distribution at the whole plant level. The plant hormone-abscisic acid (ABA) regulates many plant responses to environmental stimuli (Raghavendra et al. 2010, Seo et al. 2012). Many ABA-regulated genes, such as the ABFs (Choi et al. 2000), CBL9 (Pandey et al. 2004), ABR1 (Kim et al. 2003) and CIPK3 (Pandey et al. 2005), have been identified in plant tolerance abiotic stresses. At present, many researches indicates that ABA is closely associated with sugar signaling in plants (Rook et al. 2006, Dekkers et al. 2008). For example, genetic analysis reveals that several ABA regulated genes and transcription factors are induced by sugar signals, such as HXK1, HXK2, ABI3, ABI4, ABI5, ABF2, ABF3 and ABF4 (Rolland et al. 2006, Dekkers et al. 2008, Hanson and Smeekens 2009). Mutant analysis showed that 3 sucrose response mutants, including sucrose uncoupled (sun), impaired sucrose induction (isi) and sugar insensitive (sis), were ABA deficient mutants (i.e. aba2/isi4/sis4 and aba3/gin5) and ABA insensitive4 (abi4/sun6/isi3/sis5), which were identified as sugar insensitive (Arenas-Huertero et al. 2000, Huijser et al. 2000, Laby et al. 2000, Rook et al. 2001). Moreover, ABA can regulate the process of sucrose loading and unloading (Tanner et al. 1980, Wyse et al. 1980, Berüter 1983, Vreugdenhil 1983, Schussler et al. 1984). Recently, Peng et al. (2011) found that MdSUT1 (Malus pumila Mill.) in apple fruit may be a component of ABA signaling pathways associated with the regulation of photoassimilated transport. However, molecular genetic evidence linking sucrose transporter with ABA-regulated biological functions under abiotic stresses has been lacking (Peng et al. 2011). We have identified AtSUC4 mutants that had higher sucrose, fructose and glucose in shoots and lower those in roots, as compared to that in WT, in salt and drought stresses (Gong et al. 2013a, b). It is suggested that AtSUCs play an important role in mediating sugar distribution in abiotic stress. To systematically understand how AtSUCs regulate sucrose distribution in abiotic stress, we carried out an experiment to analyze gene expression of the AtSUC family by public microarray data and qRT-PCR in plants exposed to abiotic stresses. We found that AtSUC2 and AtSUC4 are clearly induced in response to salt, osmotic, drought, low temperature and exogenous ABA treatments. This article is protected by copyright. All rights reserved
Mutation of AtSUC2 and AtSUC4 affects the sucrose distribution in shoots and roots, resulting in hypersensitivity of seed germination and seedling growth to abiotic stresses and to exogenous ABA treatments. The function of AtSUC2 and AtSUC4 in stress responses may involve in an ABF-dependent ABA signaling pathway and a sucrose signaling pathway. These findings suggest that AtSUC2 and AtSUC4 are important regulators of the responses of Arabidopsis to abiotic stresses and to ABA, and that ABA can regulate sucrose transport and sucrose balance in the plant by controlling AtSUC2 and AtSUC4, thereby promoting plant stress tolerance.
Materials and methods The expression of AtSUCs analyzed by public microarray data The expression of all genes in Arabidopsis under control and stress treatments was available in a public microarray data set, WeigelWorld (http://weigelworld.org/resources/microarray/AtGenExpress) (Schmid et al. 2005). Experimental statistical analyses were performed by the original authors or the database providers. The expression of AtSUCs was determined at different stress treatments (salt, mannitol, drought and cold) at 0, 0.5, 1, 3, 6, 12 and 24 h, or different hormone treatments at 0, 0.5, 1 and 3 h, respectively (Schmid et al. 2005). We calculated the ratio of AtSUCs expression in the treated plants to that in the control plants (setting the control value to 1) to identify the key AtSUCs that responded to the different stress and hormone treatments.
The expression of genes analyzed by qRT-PCR To assay the expression levels of key AtSUCs genes (AtSUC2 and AtSUC4) in WT after stress and ABA treatments, real-time quantitative reverse transcription PCR (qRT-PCR) was performed with the RNA samples isolated from 14-day-old seedlings at the indicated times after the different treatments. We adopted the consistent treatment methods with public microarray data. For drought stress, the plants were stressed by 15 min dry air stream (clean bench) until 10% loss of fresh weight, and then incubation in closed vessels back in the illuminating incubator. For other stresses and ABA treatment, seedlings were incubated at 4°C for cold stress, or added to a concentration of 150 mM NaCl, 300 mM mannitol, and 10 µM ABA in the solution. About 80 mg seedlings in whole plant were sampled at 0, 1, 3, 6, and 12 h time points. Total RNA was isolated with RNasy plant mini kit (Qiagen, Hilden, Germany) supplemented with an on-column DNA digestion (Qiagen RNase-Free DNase set; Hilden, Germany). A 2 µg aliquot of RNA was reverse transcribed with Superscript II RT kit (Invitrogen, USA) in a 25 µL reaction volume at 42°C for 1 h. The primers are listed in Table S10. The expression levels of AtACT1 were served as an internal control. The suitability of the oligonucleotide sequences in term of efficiency of annealing was evaluated in advance using the Primer 5.0 program. The cDNA was amplified using SYBR Premix Ex TaqTM (Takara Biotechnology Co., Japan) in 10 µL volume and analyzed using the System LightCycler480 (Roche Applied Science, Germany). The expression level of each gene was calculated by delta-delta Ct and Ramakers et al. (2003) methods. This article is protected by copyright. All rights reserved
To assay the expression levels of various ABA-responsive genes, ABF-responsive genes and ABF-regulated genes after ABA treatment in WT, AtSUC2 and AtSUC4 mutants, qRT-PCR analysis was performed with RNA samples isolated from 14-day-old seedlings harvested at 6 h after the treatments with or without 40 µM ABA. Total RNA extraction and reverse transcription were performed as described above. The primers used for qRT-PCR are listed in Table S11 and the procedures were adopted as described above. To analyze the stress and ABA-induced expression of AtSUC2 and AtSUC4 in AtSUC3 mutant, total RNA extraction, reverse transcription and qRT-PCR procedures were performed using 14-day-old Atsuc3-1 and WT seedlings harvested at 0 or 6 h. after the treatments with the different stresses and ABA. The primers used for qRT-PCR are listed in Table S11. The ratio of AtSUC2 and AtSUC4 expression was relative to the expression of AtSUC2 and AtSUC4 in Atsuc3-1 compared to the genes’ expression in WT.
Screening of T-DNA insertion mutants The T-DNA insertion lines of the AtSUC2 (At1g22710) in the Ws-2 ecotype background, AtSUC3 (At2g02860) and AtSUC4 (At1g09960) in the Col-0 ecotype background were obtained from the Arabidopsis Biological Resource Center (ABRC). The T-DNA insertion mutant’s lines include Atsuc2-1 (CS3876; Gottwald et al. 2000), Atsuc2-3 (CS3878; Gottwald et al. 2000), Atsuc3 (SALK_077715), Atsuc4-1 (SALK_100140) and Atsuc4-2 (SALK_021916). The mutant lines were genotyped by amplifying the genome with the left genomic primer (LP) and right genomic primer (RP). The primers used for this screen are listed in Table S10. The AtACT1 was used as a quantitative control. The T-DNA insertion in mutants was confirmed by PCR and DNA gel blot analysis. All of the PCR procedures were performed using PrimerSTART ® HS DNA polymerase (Takara Biotechnology Co., Japan) to enhance fidelity.
Growth conditions and phenotype analysis Plants were grown in a growth chamber at 22°C on half-strength MS medium with 0.7% (w/v) agar (pH 6) and no exogenous sucrose or 3% exogenous sucrose at about 80 μmol photons m–2 s–1 or in compost soil at 120 μmol photons m–2 s–1 at 22°C, a 16 h-light /8 h-night dark regime and 75% relative humidity. For the germination assay, about 100 seeds each from WT and mutants were sterilized and planted in triplicate on half-strength MS medium. The medium was supplemented with and or without different concentrations of NaCl (0, 75, and 100 mM), mannitol (0, 250 and 300 mM), or ABA (0, 1 and 2 µM) at 22, 14 (cold stress) or 10°C (cold stress). The germination (emergence of radicals) was scored daily at the indicated times. To assay leaf area and primary root length, seeds each from WT and mutants were planted on half-strength MS medium supplemented with different concentrations of NaCl (0, 50 and 100 mM), or mannitol (0, 250 and 300 mM), ABA (0, 3, 5 µM or 0, 0.3, 0.5 µM) at 22, 14 or 10°C. We also examined the germination and growth, when WT and mutants This article is protected by copyright. All rights reserved
were planted on MS medium supplemented with 3% exogenous sucrose in abiotic stress and ABA treatments. The seedling were investigated and photographed at 20 d. At least 20 seedlings in each experiment were observed, and each experiment was repeated three times. The manipulation and statistical analyses of data were carried out by EXCEL 2003 and SPSS statistical software 13.0.
Extraction of total soluble sugar and sucrose content determinations Because roots were changed too short in the relatively higher concentration treatments, 20-day-old seedlings from WT and mutant plants were chosen from the relatively lower treatments (75 mM NaCl, 250 mM mannitol, at 14°C and 0.3 µM ABA) to extract total soluble sugar according to Sahrawy et al. (2004). Approximately 300 seedlings (3-time replication) were examined in medium with or without exogenous sucrose in different abiotic stress and ABA treatments. The sucrose concentrations were determined using high-performance liquid chromatography (HPLC). HPLC analysis was performed on an Agilent 1100 HPLC system equipped with a refractive index detector (Agilent Technologies, Santa Clara, CA, USA). The column used was the amino kind (Teknokroma, kromasil, 100-5 NH2 15×0.46 cm2, AKZONOBEL, Sweden), thermostatized at 28°C. The flow rate was set to 1.0 mL min– 1
and injections of 20 µL were made. The ratio of acetonitrile and the deionized water used was 80%
to 20%. Sucrose concentrations were determined and quantified by comparison with known standards. Three technical replications for three biological replicates were at least performed for each sample. The numerical data were used for statistical analyses by Microsoft Excel 2003 and SPSS statistical software 13.0. * and ** indicates significant differences in comparison to WT at P
College of Biological Science and Technology, Shenyang Agricultural University, Shenyang, 100866,
China b
Corn Research Institute, Liaoning Academy of Agricultural Sciences, Shenyang 110161, China
c
College of Agriculture and Biotechnology, China Agricultural University, Beijing 100094, China
d
College of Land and Environment, Shenyang Agricultural University, Shenyang, 100866, China
*Corresponding author, e-mail: [email protected] †
These authors contributed equally to this work.
Sucrose transporters (SUCs or SUTs) play a central role, as they orchestrate sucrose allocation both intracellularly and at the whole plant level. Previously, we found AtSUC4 mutants changing sucrose distribution under drought and salt stresses. Here, we systematically examined the role of Arabidopsis AtSUC2 and AtSUC4 in response to abiotic stress. The results showed that significant induction of AtSUC2 and AtSUC4 in salt, osmotic, low temperature and exogenous ABA treatments by public microarray data and real-time quantitative reverse transcription PCR (qRT-PCR) analyses. The loss-of-function mutation of AtSUC2 and AtSUC4 led to hypersensitive responses to abiotic stress and ABA treatment in seed germination and seedling growth. These mutants also showed higher sucrose content in shoots and lower sucrose content in roots, as compared to that in wild-type plants, and inhibited the ABA-induced expression of many stress-responsive genes and ABA-responsive genes, especially ABFs and ABF-downstream and upstream genes. The loss-of-function mutant of AtSUC3, a unique putative sucrose sensor, reduced the expression of AtSUC2 and AtSUC4 in response to abiotic stresses and ABA. These findings confirmed that AtSUC2 and AtSUC4 are important regulators in plant abiotic stress tolerance by the use of an ABA signaling pathway, which may be crossed with sucrose signaling.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ppl.12225 This article is protected by copyright. All rights reserved
Abbreviations – Abscisic acid, ABA; ABSCISIC ACID-INSENSITIVE2, ABI2; Arabidopsis Biological Resource Center, ABRC; ABA repressor 1, ABR1; High-performance liquid chromatography, HPLC; Impaired sucrose induction, isi; Left genomic primer, LP; Right genomic primer, RP; Real-time quantitative reverse transcription PCR, qRT-PCR; Reverse transcription PCR, RT-PCR; Sucrose, Suc; Sieve elements, SEs; Sugar insensitive, sis; Sucrose non-fermenting 1-related protein kinase, SnRKs; Sucrose phosphate synthase, SPS; Sucrose synthase, SuSy; Sucrose transporters, SUCs or SUTs; Wild type, WT
Introduction Plant growth and productivity are greatly affected by environmental stresses such as high salinity, dehydration, and low temperature. The responses and adaptations of plants to these stresses can occur at the molecular, cellular, physiological, and biochemical levels (Chinnusamy et al. 2005, Urano et al. 2010, Fujii and Zhu 2012). As a metabolite, sucrose plays an important role in stress tolerance, serving as an osmolyte, a signaling molecule and/or a nutritional substance (Yu 1999, Gupta and Kaur 2005, Ruan et al. 2010). The expressions of some sucrose related genes, such as SuSy (sucrose synthase) (Baud et al. 2004), SPS (sucrose phosphate synthase) (Pelleschi et al. 1997) and SnRK2s (sucrose non-fermenting 1-related protein kinase2) (Fujii and Zhu 2012, Zhang et al. 2011) have been reported to change in response to abiotic stresses. Sucrose transporters (SUCs or SUTs) are an important exporter of photosynthetically produced sugar, principally sucrose, from higher plant source leaves to sink tissues. In Arabidopsis, 9 AtSUCs have been identified and researched on some of their expressions and functions. AtSUC2, is expressed in companion cells collection and transport phloem in source leaves (Truernit and Sauer 1995, Martens et al. 2006) and functions in loading sucrose into the phloem sieve elements (SEs), as determined by tissue-specific complementation of different promoters (Srivastava et al. 2008, 2009a) and by
14
C labelling studies (Gottwald et al. 2000). This protein is also found within the vascular
bundles of sink tissues such as stamens, siliques, and roots (Truernit and Sauer 1995, Stadler and Sauer 1996), suggesting a role in the retrieval of sucrose that leaks out of sieve elements during long-distance transport (Stadler and Sauer 1996). When planted without sucrose, Atsuc2 mutant seedlings are smaller than wild-type (WT) seedlings (Gottwald et al. 2000). AtSUC4 and AtSUC3 are also expressed in minor veins of source leaves of mature plants (Weise et al. 2000, Barker et al. 2000, Aoki et al. 2012), suggesting an involvement in phloem loading of sucrose in source organs. The sucrose/proton symporter, AtSUC4, is also expressed in vacuoles and most likely regulates the transport and vacuolar storage of photosynthetically derived sucrose (Endler et al. 2006, Schulz et al. 2011).
This article is protected by copyright. All rights reserved
Sporadic reports have appeared regarding the role of SUTs in plant responses to abiotic stresses. The expression of AgSUT1, which codes for the high affinity of AgSUT1 sucrose/H+ transporter in celery (Apium graveolens), was decreased in all plant organs in response to salinity stress—a more marked decrease in roots may have reflected a decreased metabolic demand for sucrose in response to stress (Noiraud et al. 2000). Among the five sucrose transporters identified in rice (Oryza sativa cv. Nipponbare), only OsSUT2 expression was up-regulated following exposure to drought and salinity treatments (Ibraheem et al. 2011, Lundmark et al. 2006) found a low temperature induced up-regulation of AtSUC1 and AtSUC2 when analyzing the role of the sucrose-phosphate synthase (SPSox) in carbon partitioning of Arabidopsis at low temperature. Recent studies indicate that Aspen (Populus tremula) transformed with PtaSUT4-RNAi wilt when exposed to a short-term drought, indicating a role for PtaSUT4 in drought tolerance (Frost et al. 2012). Although these results indicate that SUCs are involved in plant response abiotic stress, it is not clear that the potential stress tolerance functions of the SUCs that control SUC distribution at the whole plant level. The plant hormone-abscisic acid (ABA) regulates many plant responses to environmental stimuli (Raghavendra et al. 2010, Seo et al. 2012). Many ABA-regulated genes, such as the ABFs (Choi et al. 2000), CBL9 (Pandey et al. 2004), ABR1 (Kim et al. 2003) and CIPK3 (Pandey et al. 2005), have been identified in plant tolerance abiotic stresses. At present, many researches indicates that ABA is closely associated with sugar signaling in plants (Rook et al. 2006, Dekkers et al. 2008). For example, genetic analysis reveals that several ABA regulated genes and transcription factors are induced by sugar signals, such as HXK1, HXK2, ABI3, ABI4, ABI5, ABF2, ABF3 and ABF4 (Rolland et al. 2006, Dekkers et al. 2008, Hanson and Smeekens 2009). Mutant analysis showed that 3 sucrose response mutants, including sucrose uncoupled (sun), impaired sucrose induction (isi) and sugar insensitive (sis), were ABA deficient mutants (i.e. aba2/isi4/sis4 and aba3/gin5) and ABA insensitive4 (abi4/sun6/isi3/sis5), which were identified as sugar insensitive (Arenas-Huertero et al. 2000, Huijser et al. 2000, Laby et al. 2000, Rook et al. 2001). Moreover, ABA can regulate the process of sucrose loading and unloading (Tanner et al. 1980, Wyse et al. 1980, Berüter 1983, Vreugdenhil 1983, Schussler et al. 1984). Recently, Peng et al. (2011) found that MdSUT1 (Malus pumila Mill.) in apple fruit may be a component of ABA signaling pathways associated with the regulation of photoassimilated transport. However, molecular genetic evidence linking sucrose transporter with ABA-regulated biological functions under abiotic stresses has been lacking (Peng et al. 2011). We have identified AtSUC4 mutants that had higher sucrose, fructose and glucose in shoots and lower those in roots, as compared to that in WT, in salt and drought stresses (Gong et al. 2013a, b). It is suggested that AtSUCs play an important role in mediating sugar distribution in abiotic stress. To systematically understand how AtSUCs regulate sucrose distribution in abiotic stress, we carried out an experiment to analyze gene expression of the AtSUC family by public microarray data and qRT-PCR in plants exposed to abiotic stresses. We found that AtSUC2 and AtSUC4 are clearly induced in response to salt, osmotic, drought, low temperature and exogenous ABA treatments. This article is protected by copyright. All rights reserved
Mutation of AtSUC2 and AtSUC4 affects the sucrose distribution in shoots and roots, resulting in hypersensitivity of seed germination and seedling growth to abiotic stresses and to exogenous ABA treatments. The function of AtSUC2 and AtSUC4 in stress responses may involve in an ABF-dependent ABA signaling pathway and a sucrose signaling pathway. These findings suggest that AtSUC2 and AtSUC4 are important regulators of the responses of Arabidopsis to abiotic stresses and to ABA, and that ABA can regulate sucrose transport and sucrose balance in the plant by controlling AtSUC2 and AtSUC4, thereby promoting plant stress tolerance.
Materials and methods The expression of AtSUCs analyzed by public microarray data The expression of all genes in Arabidopsis under control and stress treatments was available in a public microarray data set, WeigelWorld (http://weigelworld.org/resources/microarray/AtGenExpress) (Schmid et al. 2005). Experimental statistical analyses were performed by the original authors or the database providers. The expression of AtSUCs was determined at different stress treatments (salt, mannitol, drought and cold) at 0, 0.5, 1, 3, 6, 12 and 24 h, or different hormone treatments at 0, 0.5, 1 and 3 h, respectively (Schmid et al. 2005). We calculated the ratio of AtSUCs expression in the treated plants to that in the control plants (setting the control value to 1) to identify the key AtSUCs that responded to the different stress and hormone treatments.
The expression of genes analyzed by qRT-PCR To assay the expression levels of key AtSUCs genes (AtSUC2 and AtSUC4) in WT after stress and ABA treatments, real-time quantitative reverse transcription PCR (qRT-PCR) was performed with the RNA samples isolated from 14-day-old seedlings at the indicated times after the different treatments. We adopted the consistent treatment methods with public microarray data. For drought stress, the plants were stressed by 15 min dry air stream (clean bench) until 10% loss of fresh weight, and then incubation in closed vessels back in the illuminating incubator. For other stresses and ABA treatment, seedlings were incubated at 4°C for cold stress, or added to a concentration of 150 mM NaCl, 300 mM mannitol, and 10 µM ABA in the solution. About 80 mg seedlings in whole plant were sampled at 0, 1, 3, 6, and 12 h time points. Total RNA was isolated with RNasy plant mini kit (Qiagen, Hilden, Germany) supplemented with an on-column DNA digestion (Qiagen RNase-Free DNase set; Hilden, Germany). A 2 µg aliquot of RNA was reverse transcribed with Superscript II RT kit (Invitrogen, USA) in a 25 µL reaction volume at 42°C for 1 h. The primers are listed in Table S10. The expression levels of AtACT1 were served as an internal control. The suitability of the oligonucleotide sequences in term of efficiency of annealing was evaluated in advance using the Primer 5.0 program. The cDNA was amplified using SYBR Premix Ex TaqTM (Takara Biotechnology Co., Japan) in 10 µL volume and analyzed using the System LightCycler480 (Roche Applied Science, Germany). The expression level of each gene was calculated by delta-delta Ct and Ramakers et al. (2003) methods. This article is protected by copyright. All rights reserved
To assay the expression levels of various ABA-responsive genes, ABF-responsive genes and ABF-regulated genes after ABA treatment in WT, AtSUC2 and AtSUC4 mutants, qRT-PCR analysis was performed with RNA samples isolated from 14-day-old seedlings harvested at 6 h after the treatments with or without 40 µM ABA. Total RNA extraction and reverse transcription were performed as described above. The primers used for qRT-PCR are listed in Table S11 and the procedures were adopted as described above. To analyze the stress and ABA-induced expression of AtSUC2 and AtSUC4 in AtSUC3 mutant, total RNA extraction, reverse transcription and qRT-PCR procedures were performed using 14-day-old Atsuc3-1 and WT seedlings harvested at 0 or 6 h. after the treatments with the different stresses and ABA. The primers used for qRT-PCR are listed in Table S11. The ratio of AtSUC2 and AtSUC4 expression was relative to the expression of AtSUC2 and AtSUC4 in Atsuc3-1 compared to the genes’ expression in WT.
Screening of T-DNA insertion mutants The T-DNA insertion lines of the AtSUC2 (At1g22710) in the Ws-2 ecotype background, AtSUC3 (At2g02860) and AtSUC4 (At1g09960) in the Col-0 ecotype background were obtained from the Arabidopsis Biological Resource Center (ABRC). The T-DNA insertion mutant’s lines include Atsuc2-1 (CS3876; Gottwald et al. 2000), Atsuc2-3 (CS3878; Gottwald et al. 2000), Atsuc3 (SALK_077715), Atsuc4-1 (SALK_100140) and Atsuc4-2 (SALK_021916). The mutant lines were genotyped by amplifying the genome with the left genomic primer (LP) and right genomic primer (RP). The primers used for this screen are listed in Table S10. The AtACT1 was used as a quantitative control. The T-DNA insertion in mutants was confirmed by PCR and DNA gel blot analysis. All of the PCR procedures were performed using PrimerSTART ® HS DNA polymerase (Takara Biotechnology Co., Japan) to enhance fidelity.
Growth conditions and phenotype analysis Plants were grown in a growth chamber at 22°C on half-strength MS medium with 0.7% (w/v) agar (pH 6) and no exogenous sucrose or 3% exogenous sucrose at about 80 μmol photons m–2 s–1 or in compost soil at 120 μmol photons m–2 s–1 at 22°C, a 16 h-light /8 h-night dark regime and 75% relative humidity. For the germination assay, about 100 seeds each from WT and mutants were sterilized and planted in triplicate on half-strength MS medium. The medium was supplemented with and or without different concentrations of NaCl (0, 75, and 100 mM), mannitol (0, 250 and 300 mM), or ABA (0, 1 and 2 µM) at 22, 14 (cold stress) or 10°C (cold stress). The germination (emergence of radicals) was scored daily at the indicated times. To assay leaf area and primary root length, seeds each from WT and mutants were planted on half-strength MS medium supplemented with different concentrations of NaCl (0, 50 and 100 mM), or mannitol (0, 250 and 300 mM), ABA (0, 3, 5 µM or 0, 0.3, 0.5 µM) at 22, 14 or 10°C. We also examined the germination and growth, when WT and mutants This article is protected by copyright. All rights reserved
were planted on MS medium supplemented with 3% exogenous sucrose in abiotic stress and ABA treatments. The seedling were investigated and photographed at 20 d. At least 20 seedlings in each experiment were observed, and each experiment was repeated three times. The manipulation and statistical analyses of data were carried out by EXCEL 2003 and SPSS statistical software 13.0.
Extraction of total soluble sugar and sucrose content determinations Because roots were changed too short in the relatively higher concentration treatments, 20-day-old seedlings from WT and mutant plants were chosen from the relatively lower treatments (75 mM NaCl, 250 mM mannitol, at 14°C and 0.3 µM ABA) to extract total soluble sugar according to Sahrawy et al. (2004). Approximately 300 seedlings (3-time replication) were examined in medium with or without exogenous sucrose in different abiotic stress and ABA treatments. The sucrose concentrations were determined using high-performance liquid chromatography (HPLC). HPLC analysis was performed on an Agilent 1100 HPLC system equipped with a refractive index detector (Agilent Technologies, Santa Clara, CA, USA). The column used was the amino kind (Teknokroma, kromasil, 100-5 NH2 15×0.46 cm2, AKZONOBEL, Sweden), thermostatized at 28°C. The flow rate was set to 1.0 mL min– 1
and injections of 20 µL were made. The ratio of acetonitrile and the deionized water used was 80%
to 20%. Sucrose concentrations were determined and quantified by comparison with known standards. Three technical replications for three biological replicates were at least performed for each sample. The numerical data were used for statistical analyses by Microsoft Excel 2003 and SPSS statistical software 13.0. * and ** indicates significant differences in comparison to WT at P
