The Arabidopsis J Protein AtJ1 is Essential for Seedling Growth, Flowering Time Control and ABA Response Min Young Park and Soo Young Kim* *Corresponding author: E-mail, [email protected]; Fax, +82-62-530-2169. (Received May 7, 2014; Accepted October 8, 2014)
We describe the in planta function of an Arabidopsis J protein gene, AtJ1. We isolated an ABA-hypersensitive mutant, named as793 (ABA-hypersensitive 793), by activation tagging screen. Analysis of the mutant revealed that T-DNA was inserted into the gene encoding AtJ1, thereby abolishing its expression. as793 plants grew very poorly under normal growth conditions; their seed setting efficiency was lower and their flowering was delayed compared with wild-type plants. Moreover, as793 plants were ABA hypersensitive and drought tolerant. In parallel analyses, we found that another AtJ1 knockout mutant acquired from the Arabidopsis Stock Center exhibited the same phenotypes as as793 and that its phenotypes could be complemented by the wild-type AtJ1. At the molecular level, we found that the expression of a large number of genes involved in embryogenesis, flowering time control and stress response was altered in as793. Others previously reported that AtJ1 is a mitochondrial protein involved in thermotolerance. Our results further indicate that AtJ1 is essential for normal plant growth, from embryogenesis to flowering and seed setting. Additionally, the ABA hypersensitivity of as793 suggests that AtJ1 may function as a negative regulator of ABA response. Keywords: ABA Abiotic stress Chaperone Flowering Hsp70 J protein. Abbreviations: ER, endoplasmic reticulum; Hsp70, heat shock protein 70; KO, knockout; MS. Murashige and Skoog; NEF, nucleotide exchange factor; RT–PCR, reverse transcription–PCR; TAIL-PCR, thermal asymmetric interlaced PCR.
Introduction Hsp70 family proteins (i.e. 70 kDa heat shock proteins; Hsp70s) are ubiquitous molecular chaperones that function in a variety of cellular processes (Kampinga and Craig 2010, Hartl et al. 2011). Under normal conditions, they facilitate the folding of nascent polypeptides, protein transport across cell membranes and oligomeric protein assembly. Under stress conditions, Hsp70s help refolding or degradation of stress-denatured proteins. The functions of Hsp70s depend on their binding and release of substrate proteins in an ATP-dependent cycle (Kampinga and Craig 2010, Hartl et al. 2011). However, Hsp70s do not
function alone. They require another class of chaperones, J protein/Hsp40 family members, and nucleotide exchange factors (NEFs) for their full activities. In the canonical mode of Hsp70 action, a J protein binds to a substrate protein and subsequently interacts with an ATP-bound Hsp70, delivering the substrate protein to the Hsp70–ATP complex. The binding of J protein and substrate stimulates the ATPase activity of Hsp70 and hydrolysis of the bound ATP, thereby causing conformational change and tighter binding of the substrate to the resulting ADP–Hsp70 complex. The J protein is then released from the complex, and ADP is exchanged with ATP by an NEF. Hsp70s have low affinity for substrates in their ATP-bound state. Thus, the nucleotide exchange is followed by substrate release, and a new cycle begins. In plants and other organisms, there are more J proteins than Hsp70s and NEFs (Kampinga and Craig 2010, Hartl et al. 2011). In Arabidopsis, for example, the Hsp70 family consists of 14 members, whereas the J protein family consists of at least 105 members (Lin et al. 2001, Sung et al. 2001, Finka et al. 2011). The functional diversity of the Hsp70 machinery is therefore determined, in major part, by J proteins. J proteins possess a highly conserved signature domain, which was originally found in the prototype J protein Escherichai coli DnaJ and thus named J domain (Kampinga and Craig 2010). The J domain consists of approximately 70 amino acids and contains an invariable His-Pro-Asp (HPD) tripeptide motif. The HPD motif is essential for the stimulation of ATPase activity of Hsp70s. Although they possess the highly conserved J domain, J proteins are diverse in their overall structure and grouped into three classes: Type I, II and III, or Type A, B and C. Type I J proteins have a domain structure similar to that of DnaJ, i.e. the N-terminal J domain is followed by a glycine/phenylalanine-rich region (G/F region), a zinc-finger motif (CXXCXGXG) and a C-terminal extension domain. Type II J proteins possess a J domain and a G/F region but lack the zinc-finger domain. Other J proteins belong to Type III. In Arabidopsis, Type III is the most abundant class (approximately 90 members) (Miernyk 2001, Rajan and D’Silva 2009, Finka et al. 2011, Chiu et al. 2013). Several different systems have been proposed for the nomenclature of Arabidopsis J proteins (Miernyk 2001, Rajan and D’Silva 2009, Finka et al. 2011). J proteins are localized not only in the cytosol but also in organelles such as the mitochondria, chloroplast, endoplasmic reticulum (ER) and nucleus (Miernyk 2001, Rajan and D’Silva
Plant Cell Physiol. 55(12): 2152–2163 (2014) doi:10.1093/pcp/pcu145, Advance Access publication on 13 October 2014, available online at www.pcp.oxfordjournals.org ! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
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Regular Paper
Departments of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, South Korea
Plant Cell Physiol. 55(12): 2152–2163 (2014) doi:10.1093/pcp/pcu145
Results Isolation of the ABA-hypersensitive mutant as793 We carried out an activation tagging screen to isolate ABA response mutants. A mutant library consisting of approximately 25,000 independent transgenic lines was prepared and screened for the isolation of mutants with altered ABA sensitivity, as described elsewhere (Park et al. 2011). A number of mutants were isolated, and one of them, designated as793 (ABA-hypersensitive 793), was characterized further in this study (Fig. 1A). Thermal asymmetric interlaced PCR (TAIL-PCR) (Liu et al. 1995) was carried out to determine the T-DNA insertion site in as793, and T-DNA was found to be located in one of the introns
of a gene (At1g28210) encoding a J protein, AtJ1 (Fig. 1B) (Kroczynska et al. 1996). Subsequent reverse transcription– PCR (RT–PCR) analysis to determine the AtJ1 transcript level indicated that its expression was completely abolished in the mutant (Fig. 1C). Thus, as793 is a knockout (KO) mutant of AtJ1. AtJ1 is also known as AtDjB1 (Miernyk 2001), atDiC23 (Rajan and D’Silva 2009) and DJC20 (Finka et al. 2011).
as793 is impaired in seedling growth and seed setting One of the most distinct phenotypes of as793 was poor seedling growth, i.e. the mutant seedlings grew very slowly compared with wild-type plants (Fig. 1D). To confirm that the phenotype is the loss-of-function phenotype of AtJ1, we acquired its KO mutant atj1 (SALK_049553), and, after the confirmation of T-DNA insertion in the annotated site, we analyzed its phenotypes in parallel with as793. AtJ1 expression is abolished completely in atj1 (Fig. 1C). Additionally, we prepared and analyzed AtJ1 complementation lines by transforming atj1 with a genomic DNA fragment containing the wild-type AtJ1 coding region and its promoter (Fig. 2). Like as793, atj1 seedlings also grew very slowly (Figs. 1D, 2). Consequently, both as793 and atj1 plants took longer than wild-type plants to reach the bolting stage and maturity (see below). In contrast, the growth of the complementation line was normal, indicating that the poor seedling growth is the KO phenotype of AtJ1. Another noticeable phenotype of as793 and atj1 was poor seed setting efficiency. As shown in Fig. 3A and B, the mutant plants had fewer siliques than wild-type plants and, frequently, silique formation was not complete. Close examination of the aberrant siliques revealed that they carried fewer seeds than wild-type siliques. Many aborted seeds, i.e. seeds which failed to develop, were observed, and, occasionally, partially developed seeds were found in the siliques (Fig. 3B). Thus, seed setting efficiency is impaired in the AtJ1 mutants.
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2009, Finka et al. 2011). As co-chaperones of Hsp70s, J proteins are involved in various cellular processes mentioned above. However, the specific functions of most J proteins in plants are poorly understood, except for a number of cases. An Arabidopsis Type I J protein GFA2/atDjA3, which is an ortholog of the yeast mitochondrial J protein MDJ1, is required for synergid cell death during megagametogenesis (Christensen et al. 2002). The cytosolic Type III J protein JAC1/atDjC18 is essential for phototropin-mediated chloroplast movement (Suetsugu et al. 2005). Five ER-localized J proteins, which are orthologs of yeast Sec63p, Scj1 or Jem1p, have been reported (Yamamoto et al. 2008). AtERdj2A/atDjC20, an ortholog of yeast Sec63p functioning in protein translocation across the ER membrane, is essential for seedling growth and pollen germination. AtERdj3A/atDjB9, AtERdj3B/atDjA8 and AtP58IPK/atDjC19, orthologs of yeast Scj1 and Jem1 functioning in protein folding in the ER, can partially complement the yeast jem1Dscj1 mutant. Cytoskeleton-associated Arabidopsis J proteins ARG1/atDjB11, ARL1/atDjB12 and ARL2/atDjB13 are highly homologous to each other and participate in the gravity signaling process (Sedbrook et al. 1999, Boonsirichai et al. 2003, Guan et al. 2003). The peroxisomal J protein GRV2/KAM2/ atDjC30 is involved in endosome formation and the tropic growth response (Silady et al. 2004, Tamura et al. 2007). Recently, J20/atDjC29 in plastids was shown to deliver the inactive deoxyxylulose 5-phosphate synthase enzyme to Hsp70 for proper folding or degradation (Pulido et al. 2013). In this study, we provide evidence that the Type III J protein AtJ1 is required for proper seedling growth and ABA response. In the course of our activation tagging screen to identify ABA response mutants, we isolated a mutant in which AtJ1 expression was abolished. Previously, Kroczynska et al. (1996) showed that recombinant AtJ1 stimulates the ATPase activity of both E. coli DnaK and maize endosperm Hsp70 in vitro, and the expression of the AtJ1 gene in an E. coli dnaJ deletion mutant can rescue the growth ability of E. coli under heat stress. Recently, Zhou et al. (2012) reported that AtJ1 facilitates thermotolerance by protecting cells against heatinduced oxidative damage. Here, we show that AtJ1 is essential for normal seedling growth, flowering time control and ABA response.
Flowering of as793 is delayed Although as793 and atj1 seedlings grew slowly and their seed setting efficiency was low, they grew to maturity, and mature atj1 and as793 plants were similar to wild-type plants in size and stature (Fig. 2D, E). However, it took longer for the mutant plants to reach the bolting stage (Fig. 3C). Under our growth conditions (i.e. 16 h light/8 h dark cycle), wild-type (Col-0) plants started bolting approximately 23 d after seed sowing, whereas the mutant plants bolted 32 d after sowing (Fig. 3D, E). Late bolting of as793 and atj1 plants is expected because the mutant seedlings grew slowly. However, it is also possible that flowering time might be delayed in the mutants. To address this possibility, we counted the number of rosette leaves at the bolting stage. Fig. 3D and E shows that the average number of rosette leaves of the wild-type plants and the complementation line was 13 at the bolting stage. In contrast, the number of rosette leaves of the mutant plants was 16 at the bolting stage. The result indicates that flowering time is delayed in the mutants. 2153
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as793 is ABA-hypersensitive as793 was isolated based on its ABA hypersensitivity during early seedling growth (Fig. 1). To better understand the ABA sensitivity of as793, we investigated ABA-associated phenotypes at various developmental stages. First, we examined the ABA sensitivity during seed germination. Mature, dry seeds were plated on Murashige and Skoog (MS) medium containing various concentrations of ABA, and rates of germination (i.e. radicle emergence) were scored daily. In the medium lacking ABA, it took approximately 2 d for approximately 80% of the as793 and atj1 seeds to germinate, whereas nearly 100% of the wild-type and the complementation line seeds germinated within 1 d after imbibition (Fig. 4A). In the presence of increasing concentrations of ABA, germination rates of as793 and atj1 seeds decreased gradually, and their germination rates were approximately 30% at 1 mM ABA under our experimental conditions (Fig. 4B). Under the same conditions, germination of the wild-type and complementation line seeds was not noticeably affected, indicating that germination of the mutant seeds was more severely affected by ABA. We next examined the ABA sensitivity during the seedling establishment stage, by determining the effect of ABA on the 2154
cotyledon greening process (Fig. 4C, D). After imbibition, seeds were plated on MS medium containing various concentrations of ABA, and seedlings with green cotyledons were counted after prolonged time (i.e. 10 d) to allow full germination. Fig. 4C and D shows that wild-type and complementation line seeds germinated and all the seedlings developed to have green cotyledons at all concentrations of ABA tested. However, the cotyledon greening efficiency of as793 and atj1 seedlings decreased rapidly due to ABA, and few seedlings developed to have green cotyledons at ABA concentrations >0.5 mM. Similarly, we investigated the ABA effect on primary root elongation. Seeds were germinated on MS medium, and germinated seedlings were transferred to media containing various concentrations of ABA. Root growth was then measured 4 d after the transfer. Fig. 4E shows that root growth of the atj1 and as793 mutant plants was more severely inhibited by ABA than that of the wild-type plants at all doses of ABA. Root growth rates of the complementation line were similar to those of the wild-type plants. Thus, AtJ1 KO mutants were ABA-hypersensitive during seed germination and post-germination growth.
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Fig. 1 Isolation of the ABA-hypersensitive mutant as793. (A) ABA sensitivity of activation-tagged mutants. Seeds from putative ABA response mutants including as793 were germinated and grown for 6 d in MS medium (MS) or MS medium with ABA (0.5 mM). (B) T-DNA insertion sites in as793 and atj1 (SALK_049553) are shown schematically. LB indicates the border sequence. Filled and open boxes indicate the exons and untranslated regions, respectively. (C) AtJ1 transcript levels were determined by semi-quantitative RT–PCR. Actin-1 was used as a reference gene. (D) Mutant seedlings grown for 10 d are shown.
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Fig. 2 AtJ1 complementation lines and growth of the atj1 and as793 mutant. (A) Growth of the wild type (Col-0), atj1 and complementation lines (atj1/AtJ1). Plants were grown in MS medium for 10 d. (B) Expression levels of AtJ1 relative to the wild-type level in the complementation lines are shown. (C) Growth of wild-type, atj1, as793 and atj1/AtJ1 (#10) plants. Plants were grown in soil for 3 (top), 5 (middle) or 6 (bottom) weeks in soil. (D, E) Fresh weight of shoot (C) or root (D) from plants grown for the indicated times. Bars represent the SD (n = 15 each).
The hypersensitivity of as793 is ABA-specific Our results indicated that germination and seedling growth of as793 and atj1 were hypersensitive to ABA. To address whether the altered hormone response was specific to ABA or not, we examined the effects of other hormones on the growth of the mutant plants. Seeds were germinated and grown in media containing various hormones or hormone precursors, such as ABA, 1-aminocyclopropane-1-caboxylic acid (ACC), benzyladenine (BA), brassinolide (BL), GA3 or IAA. Under our experimental condition, GA3 and auxin had little effect on seedling growth, whereas ACC, BA and BL had inhibitory effects. However, these hormones affected mutant seedlings and wild-type plants to similar degrees, and the hypersensitivity was observed only with ABA (Supplementary Fig. S1). The result suggests that hypersensitive response is ABA-specific.
as793 is hypersensitive to glucose and high osmolarity during early seedling growth The endogenous ABA level is increased by glucose, and ABA plays a positive role in glucose-mediated inhibition of seedling establishment, i.e. cotyledon expansion and greening (Leon and
Sheen 2003). To investigate whether AtJ1 is involved in the process, we examined the glucose effect on the cotyledon greening process. Fig. 5A and B shows that growth of the mutants was more severely inhibited by glucose than that of wildtype plants. For example, shoot growth of the mutants was almost completely inhibited at 3% glucose. On the other hand, wild-type and complementation line plants were not affected by the low concentration of glucose, and approximately 70% of their seedlings still turned green at 5% glucose. It has been well established that ABA mediates the salt response (Xiong and Zhu 2002). To determine whether AtJ1 contributes to the salt response, we examined the salt effect on germination and post-germination growth of as793. As shown in Fig. 5C, salt inhibition of germination was more severe in the atj1 and as793 mutant plants than in the wild-type plants. For example, germination of the wild-type and complementation line seeds was not affected noticeably up to 125 mM NaCl under our experimental conditions, but germination rates of the mutant seeds were reduced from 93% to approximately 25%. Similarly, the cotyledon greening process of the mutant seedlings was more severely inhibited by salt than that of wildtype and complementation line plants (Fig. 5D, E). We also 2155
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investigated the effect of high osmolarity on seed germination and cotyledon greening and observed a hypersensitive response during both germination and the cotyledon greening process (Supplementary Fig. S2). Collectively, these results indicate that as793 and atj1 mutants are hypersensitive to salt and high osmolarity during the early seedling growth stage.
as793 is drought-tolerant Our results indicated that AtJ1 is required for the normal ABA response. Because ABA controls response to abiotic stresses such as drought and high salinity (Xiong et al. 2002, Finkelstein 2013), we investigated whether drought tolerance was affected in as793 and atj1. Seedlings of similar developmental stages, i.e. 2-week-old wild-type or complementation line plants and 3-week-old as793 and atj1 plants, were subjected 2156
to water deficit conditions by withholding water for 14 d. The plants were re-watered at the end of the treatment, and survival rates were scored 3 d after re-watering. Fig. 6 shows that survival rates of as793 and atj1 plants were higher than those of the wild-type and complementation line plants: The survival rates of as793 and atj1 plants were approximately 80%, whereas the survival rates of wild-type and complementation line (atj1/ AtJ1) plants were approximately 30%. The result indicates that AtJ1 mutants are drought-tolerant, and thus suggests that AtJ1 is a negative regulator of the water-deficit response. To address the possible mechanism of the enhanced drought response, we determined the transpiration rates using detached leaves. Fig. 6C shows that the water loss rates of mutant leaves were lower than those of the wild-type or complementation line leaves, indicating that lower
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Fig. 3 Silique formation and flowering of AtJ1 mutants. (A) Siliques of 47-day-old plants. (B) Seed development in wild-type, atj1 and as793 siliques. White arrows, undeveloped ovules; white stars, aborted seeds. (C) Wild-type (Col-0), atj1, as793 and atj1/AtJ1 (#10) plants grown for 4 (top panel) or 5 (bottom panel) weeks. (D, E) Bolting time and the number of leaves in wild-type (Col-0), atj1, as793 and atj1/AtJ1 (#10) plants at the bolting stage. Plants were grown in long-day conditions, and the numbers on the y-axis represent days or leaf numbers. The data represent mean values and the small bars indicate the SE (n = 15).
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Fig. 4 ABA sensitivity of AtJ1 mutants. (A) Germination of AtJ1 mutants. Germination rates of wild-type and mutant seeds were scored in the medium lacking ABA. Experiments were carried out in triplicate (n = 50 each). (B) ABA dose–response of germination. Germination of the wild type, atj1, as793 and the complementation line (atj1/AtJ1 #10) on medium containing various concentrations of ABA was scored 3 d after imbibition. Experiments were carried out in triplicate (n = 50 each), and the small bars indicate the SE. (C) ABA dose–response of shoot development of the wild type, atj1, as793 and atj1/AtJ1 (#10). Seeds were germinated and grown for 10 d in media containing various concentrations of ABA before photographs were taken. (D) Cotyledon greening efficiency of the seedlings in (B). Experiments were carried out in triplicate (n = 50 each), and the small bars indicate the SE. (E) Root elongation assay. Seedling were germinated and grown in a medium lacking ABA for 3 d, and the seedlings were transferred to a medium containing various concentrations of ABA. Root elongation was measured 4 d after the transfer. Experiments were carried out in triplicate (n = 6 each), and the small bars indicate the SE.
transpiration rates of the mutant plants might have contributed to the enhanced drought tolerance of as793 and atj1. As mentioned earlier, the ABA level increases under water-deficit conditions, and several osmolytes, especially proline (Kishor
1995), are known to accumulate to enhance osmotolerance. Therefore, we determined their contents in as793 and found that, although ABA contents did not change noticeably, proline contents were higher in as793 than in wild-type plants 2157
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(Supplementary Fig. S3). Consistent with this observation, the expression level of the proline biosynthetic gene P5CS1 (Yoshiba et al. 1995) under normal conditions was higher (i.e. approximately three times) in as793 compared with the wildtype level (Supplementary Fig. S3). Under high salt conditions, the P5CS1 level in as793 was similar to the wild-type level. We also observed an increase in the expression level of the proline catabolic gene ProDH (Kiyosue et al. 1996) under high salt 2158
conditions (Supplementary Fig. S3). Thus, our results indicate that AtJ1 affects the expression of proline metabolic genes.
The expression of genes involved in embryogenesis, flowering time control and stress response is altered in as793 To address the possible mode of AtJ1 function, we examined whether the expression of various genes associated with the
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Fig. 5 Glucose and salt sensitivities of AtJ1 mutants. (A) Glucose dose–response of shoot development. Seeds of the wild type (Col-0), atj1, as793 and atj1/AtJ1 (#10) were germinated and grown for 10 d in media containing various concentrations of glucose. (B) Cotyledon greening efficiency of the seedlings in (A). Experiments were carried out in triplicate (n = 50 each), and the small bars indicate the SE. (C) Salt dose–response of germination. Seed germination was scored 3 d after imbibition on media containing various concentrations of salt (NaCl). Experiments were carried out in triplicate (n = 50 each), and the small bars indicate the SE. (D) Salt dose–response of shoot development. Seeds were germinated and grown for 10 d in media containing various concentrations of salt (NaCl). (E) Cotyledon greening efficiency of the seedlings in (C). Experiments were carried out in triplicate (n = 50 each), and the small bars indicate the SE.
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Fig. 6 Drought tolerance of AtJ1 mutants. (A) Water was withheld from plants of similar developmental stages (top panel) for 14 d and then the plants were watered. The picture shows the plant 3 d after the re-watering. (B) Survival rates. The values represent the mean of three independent experiments (n = 20 each), and the small bars represent the SE. (C) Transpiration rates. Leaves were detached from plants of a similar developmental stage (i.e. 4th and 5th true leaves from 21-day-old Col-0 or atj1/AtJ1 (#10) plants and 31-day-old as793 or atj1 plants) and leaf weight was measured at the indicated time interval. The values indicate weight relative to initial weight. Experiments were carried out in triplicate (n = 10 each).
phenotypes described above was altered. First, we compared the expression levels of a number of embryogenesis-related genes in the mutants with their wild-type levels by carrying out real-time RT–PCR. Fig. 7A shows that the expression of a number of genes playing important regulatory roles during embryogenesis (Capron et al. 2009, De Smet et al. 2010) was altered. WOX2, which regulates apical pattering during the early embryogenesis stage, was up-regulated approximately 3fold in as793 and atj1. On the other hand, several other genes, such as PIN1, MP/ARF5, BSK12/SSP and STM, were significantly down-regulated. Next, we determined the expression levels of flowering time control genes. As shown in Fig. 7B, the transcript level of the floral repressor FLC in the mutants was approximately three times the wild-type level. In contrast, expression levels of several positive regulators, such as FT, LFY and SOC1, were much lower (10–20%) than their wild-type levels. We also determined the expression levels of ABA- and stress-regulated genes (Fig. 7C). Among the known ABA signaling components, the expression level of ABI5, which is one of the key positive regulators of ABA response (Finkelstein 2013), was approximately 2.5 times the wild-type level in as793 and atj1. Our result further showed that a number of
stress-regulated, in particular, cold-regulated, genes were down-regulated in the mutants (Fig. 7C).
Discussion J proteins function as co-chaperones of Hsp70s by activating the ATPase activity of Hsp70s and/or by delivering substrates to Hsp70s. As mentioned in the Introduction, a number of Arabidopsis J proteins have been reported, and these studies have shown that they are involved in various cellular processes, such as gravity and tropic responses, light-dependent chloroplast movement, gametogenesis, pollen germination, protein translocation and maintenance of proper enzyme function. Considering the large number of Arabidopsis J proteins, however, only a fraction of them have been reported so far, and the specific functions of most J proteins remain to be determined. AtJ1 is a Type III Arabidopsis J protein. The gene encoding AtJ1 is constitutively expressed in various tissues and is heatinducible (Zhou et al. 2012). Previously, Kroczynska et al. (1996) showed that recombinant AtJ1 can stimulate the ATPase activity of HsP70s and is imported into mitochondria. Zhou et al. 2159
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Fig. 7 Transcript level changes in AtJ1 mutants. (A) Transcript levels of embryogenesis-related genes relative to the wild-type levels are shown: WOX2 (At5g59340), PIN1 (At1g73590), ARF5 (At1g19850), BSK12/SSP (At2g17090) and STM (At1g62360). (B) Relative expression levels of flowering time control genes: FLC (At5g10140), FT (At1g65480), LFY (At5g61850) and SOC1 (At2g45660). (C) Relative induction levels of ABA/stress-responsive genes: ABI5 (At2g36270), CBF1 (At4g25490), CBF2 (At4g25470), CBF3 (At4g25480), COR15A (At2g42540) and RAB18 (At5g66400). For (A) and (B), RNA was isolated from plants grown under normal conditions. For (C), RNA was isolated from plants subjected to low temperature (0 C) for 3 h, except for ABI5 and their relative induction levels compared with untreated levels are shown. Real-time PCRs were done in duplicate, and the small bars indicate the SE.
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Further expression analyses showed that the expression of additional genes involved in plant development and stress response was altered in as793 (Supplementary Figs. S3–S5). These include a number of genes regulating embryogenesis (ABCB1, PID, SHR, etc.) (Supplementary Fig. S5A) and seed dormancy/germination (SOMNUS and GA3ox1) (Supplementary Fig. S5B). Changes in the expression levels of stress-responsive genes were also detected (Fig. 7C; Supplementary Figs. S3, S4). In particular, RD20 expression was up-regulated 3- to 5-fold in as 793 (Supplementary Fig. S4). RD20 expression is induced by ABA and abiotic stresses, and it has been demonstrated to mediate ABA response and drought tolerance (Aubert et al. 2010, Blee et al. 2014). Thus, AtJ1 affects the expression of a large number of genes that regulate plant development and stress response. In summary, our results show that, in addition to its function in mitochondria, AtJ1 is involved in other cellular processes that affect plant development, ABA response and stress tolerance. At present, the putative substrate proteins of AtJ1, which are responsible for these cellular activities, are not known, and the mechanism of AtJ1 function remains to be determined. However, our data strongly suggest that the function of AtJ1 entails the modulation of gene expression.
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(2012) recently reported that AtJ1 functions with a mitochondrial Hsp70 to protect cells against heat-induced oxidative damage by maintaining cellular redox homeostasis. Our results further indicate that AtJ1 is essential for normal plant growth, i.e. its mutants are defective in various aspects of plant growth: embryogenesis, seed germination, seedling growth, transition to flowering and seed setting. AtJ1 KO mutants, as793 and atj1, grew very poorly, especially during the early stage of seedling growth (Figs. 1, 2). Both root and shoot growth of seedlings was slow and, as a consequence, it took much longer (approximately 9 d) for the mutant seedlings to reach the bolting stage (Fig. 3). However, plant morphology was normal and mature mutant plants were of the same size as the wild-type plants (Fig. 2), indicating that the developmental process itself was not affected in the mutants. In addition to slow seedling growth, transition to the flowering stage was also delayed: mutant plants started to bolt at the 16 leaf stage, whereas wild-type plants bolted at the 13 leaf stage. The AtJ1 mutant plants also exhibited poor seed setting efficiency. Embryo development was frequently aborted at the initial stage, silique formation was not complete and fewer siliques were observed. Furthermore, the mature embryos germinated slowly (Fig. 4A), and greater seed dormancy was observed (data not shown). In addition to general plant growth/development, the ABA response was altered in the AtJ1 mutants. The mutants were hypersensitive to ABA inhibition during germination and post-germination growth. Additionally, the mutant seedlings were hypersensitive to glucose, whose effect is mediated by ABA. Consistent with the ABA hypersensitivity, the transpiration rates of the the mutant seedlings were lower, and the mutant plants were drought-tolerant (Fig. 6). We also investigated tolerance towards other stresses and found that seedling growth was hypersensitive to high salt (Fig. 5) and high osmolarity (Supplementary Fig. S2) during seedling establishment (i.e. germination and the cotyledon greening stage). Others reported that AtJ1 is a mitochondrial protein (Kroczynska et al. 1996, Zhou et al. 2012). Thus, it can be speculated that the poor plant growth and altered ABA response may be due to the defect in the function of mitochondria, which provides the energy required for cellular activities. However, our expression analyses (Fig. 7) demonstrate that AtJ1 affects the expression of a large number of genes involved in various developmental processes. Some of the genes are regulatory genes. For instance, we observed significant downregulation of a number of genes that play important roles in embryogenesis, such as PIN1, ARF5, SSP and STM (Fig. 7A). Moreover, we observed altered expression of genes controlling flowering time. FLC, which is a repressor of flowering, was enhanced, whereas positive regulators, such as FT, LFY and SOC1, were down-regulated (Fig. 7B). This altered gene expression pattern is consistent with the late flowering phenotype of the AtJ1 mutants. Also, it is noteworthy that the expression of ABI5, which is a key ABA signaling component regulating seed dormancy, germination and early seedling growth (Finkelstein 2013), was up-regulated in as793 (Fig. 7C).
Materials and Methods Isolation of the as793 mutant The generation of activation-tagged transgenic plants and the screen for ABAhypersensitive mutants has been described elsewhere (Park et al. 2011). The TDNA insertion site in the as793 mutant was determined by TAIL-PCR (Liu et al. 1995). The T-DNA insertion site was then confirmed by sequencing the DNA fragment amplified using the T-DNA border sequence 50 -AAT AAC GCT GCG GAC ATC TA-30 and the gene-specific primer 50 -TCC ATG ATC TAC AAA AAA GCC TTG C-30 . RT–PCR analysis was performed using the primers 50 -CAA CTT TCA CTA GGG ATG GCT CA-30 and 50 -CGG AAG CGA ACA TAC TGA TCT C-30 .
Analysis of AtJ1 knockout lines The KO line (SALK_049553) of AtJ1, atj1, was obtained from the Arabidopsis Stock Center. The knockout plants were kanamycin susceptible. To purify homozygous lines, PCR was performed with genomic DNA isolated from next-generation plants grown individually. For the genotyping analysis, the primer set 50 -CAA CTT TCA CTA GGG ATG GCT CA-30 (forward), 50 -CGG AAG CGA ACA TAC TGA TCT C-30 (reverse) and 50 -CAG AAA TGG ATA AAT AGC CTT GCT T-30 (left border) was used. For semi-quantitative RT-PCR in Fig, 1, 50 -CAA CTT TCA CTA GGG ATG GCT CA-30 and 50 -CGG AAG CGA ACA TAC TGA TCT C-30 were used.
Generation of complementation lines and phenotype analysis To prepare complementation lines, a genomic DNA fragment containing the entire coding region of AtJ1 was amplified using the primer set 50 -ATG CGA AGA TTC AAC TGG GTT C-30 and 50 -cga gct cCA GCT TAA CTT GTT TGA TGT C-30 , and, after SacI digestion, cloned into AtJ1 promoter-pBI101.2. AtJ1 promoter-pBI101.2, in turn, was prepared by cloning the 1.7 kb promoter fragment amplified using the primer set 50 -gcg tcg acA GTG TCT TTA TCA TTT CGG ACA-30 and 50 -cgg gat ccG ACT CTT GTG AAT TCA AAG CTA AAC-30 into pBI101.2 and then removing the b-glucuronidase (GUS) coding region after SmaI/SacI digestion. The construct was then introduced into Agrobacterium tumefaciens strain GV3101, and the atj1 mutant
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M. Y. Park and S. Y. Kim | AtJ1 is essential for normal plant growth
RNA isolation and expression analysis Seedling RNA was isolated employing the Qiagen RNeasy plant mini kit. Seed RNA isolation was carried out employing the Sigma SpectrumTM plant total RNA kit (Sigma). For RT–PCR analysis, possible contaminating DNA was removed from RNA samples by DNase I treatment. The first-strand cDNA was synthesized using Superscript III (Invitrogen) according to the supplier’s instructions. For semi-quantitative RT–PCR, cDNA amplification was carried out within a linear range using gene-specific primers. For real-time RT–PCR, the cDNA amplification was performed with SsoFast EvaGreen supermix in a BioRad CFX96 Real-Time PCR Systems (Bio-Rad), and quantitation was carried out using the CFX96 Real-Time PCR System’s software. Actin-1 was employed as a reference gene. The primers used in the expression analysis of various genes are shown in Supplementary Tables S1 and S2.
Supplementary data Supplementary data are available at PCP online.
Funding This work was supported the Rural Development Administration, Republic of Korea [the Next Generation BioGreen 21 Program (grant PJ00819804 to S.Y.K.)]; the Ministry of Education, Science and Technology (MEST) [the Mid-career Researcher Program through an NRF grant [No. 2011-0015455 to S.Y.K.].
Acknowledgments The authors are grateful to the Kumho Life Science Laboratory of Chonnam National University for providing equipment and plant growth facilities. We thank Seul-bi Lee and Sun-geum Jeung for their technical assistance.
Disclosures The authors have no conflicts of interest to declare.
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was transformed by the vacuum infiltration method (Bechtold and Pelletier 1998). Phenotype analysis was carried out as described before (Kang et al. 2002, Kim et al. 2004). For aseptic growth, seeds were surface-sterilized by treating with 70% ethanol for 1 min and with 30% bleach for 5 min, and then washed with sterile water five times before planting. Seeds were placed at 4 C for 3–5 d and plated on MS medium solidified with 0.8% Phytoagar. The MS medium was supplemented with 1% sucrose and, when necessary, various concentrations of ABA, salt, glucose and mannitol were added to the MS medium. For the drought test, water was withheld from 9-day-old soil-grown plants until plants lost turgor completely (usually about 14 d). The treated plants were then re-watered, and survival rates were determined by counting plants that continued to grow. The same numbers of wild-type and transgenic plants were grown on the same tray to minimize experimental variation, and plants of similar developmental stages (i.e. wild-type seeds were sown 7 d later than mutant seeds) were tested.
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Sung, D.Y., Vierling, E. and Guy, C.L. (2001) Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family. Plant Physiol. 126: 789–800. Tamura, K., Takahashi, H., Kunieda, T., Fuji, K., Shimada, T. and HaraNishimura, I. (2007) Arabidopsis KAM2/GRV2 is required for proper endosome formation and functions in vacuolar sorting and determination of the embryo growth axis. Plant Cell 19: 320–332. Xiong, L., Schumaker, K.S. and Zhu, J.K. (2002) Cell signaling during cold, drought, and salt stress. Plant Cell 14 (Suppl), S165–S183. Xiong, L. and Zhu, J.K. (2002) Salt tolerance. Arabidopsis Book 1: e0048. Yamamoto, M., Maruyama, D., Endo, T. and Nishikawa, S. (2008) Arabidopsis thaliana has a set of J proteins in the endoplasmic reticulum that are conserved from yeast to animals and plants. Plant Cell Physiol. 49: 1547–1562. Yoshiba, Y., Kiyosue, T., Katagiri, T., Ueda, H., Mizoguchi, T., Yamaguchi-Shinozaki, K. et al. (1995) Correlation between the induction of a gene for delta 1-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress. Plant J. 7: 751–760. Zhou, W., Zhou, T., Li, M.X., Zhao, C.L., Jia, N., Wang, X.X. et al. (2012) The Arabidopsis J-protein AtDjB1 facilitates thermotolerance by protecting cells against heat-induced oxidative damage. New Phytol. 194: 364–378.
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Miernyk, J.A. (2001) The J-domain proteins of Arabidopsis thaliana: an unexpectedly large and diverse family of chaperones. Cell Stress Chaperones 6: 209–218. Park, M.Y., Kang, J.Y. and Kim, S.Y. (2011) Overexpression of AtMYB52 confers ABA hypersensitivity and drought tolerance. Mol. Cells 31: 447–454. Pulido, P., Toledo-Ortiz, G., Phillips, M.A., Wright, L.P. and RodriguezConcepcion, M. (2013) Arabidopsis J-protein J20 delivers the first enzyme of the plastidial isoprenoid pathway to protein quality control. Plant Cell 25: 4183–4194. Rajan, V.B. and D’Silva, P. (2009) Arabidopsis thaliana J-class heat shock proteins: cellular stress sensors. Funct. Integr. Genomics 9: 433–446. Sedbrook, J.C., Chen, R. and Masson, P.H. (1999) ARG1 (altered response to gravity) encodes a DnaJ-like protein that potentially interacts with the cytoskeleton. Proc. Natl Acad. Sci. USA 96: 1140–1145. Silady, R.A., Kato, T., Lukowitz, W., Sieber, P., Tasaka, M. and Somerville, C.R. (2004) The gravitropism defective 2 mutants of Arabidopsis are deficient in a protein implicated in endocytosis in Caenorhabditis elegans. Plant Physiol. 136: 3095–3103. Suetsugu, N., Kagawa, T. and Wada, M. (2005) An auxilin-like J-domain protein, JAC1, regulates phototropin-mediated chloroplast movement in Arabidopsis. Plant Physiol. 139: 151–162.
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We describe the in planta function of an Arabidopsis J protein gene, AtJ1. We isolated an ABA-hypersensitive mutant, named as793 (ABA-hypersensitive 793), by activation tagging screen. Analysis of the mutant revealed that T-DNA was inserted into the gene encoding AtJ1, thereby abolishing its expression. as793 plants grew very poorly under normal growth conditions; their seed setting efficiency was lower and their flowering was delayed compared with wild-type plants. Moreover, as793 plants were ABA hypersensitive and drought tolerant. In parallel analyses, we found that another AtJ1 knockout mutant acquired from the Arabidopsis Stock Center exhibited the same phenotypes as as793 and that its phenotypes could be complemented by the wild-type AtJ1. At the molecular level, we found that the expression of a large number of genes involved in embryogenesis, flowering time control and stress response was altered in as793. Others previously reported that AtJ1 is a mitochondrial protein involved in thermotolerance. Our results further indicate that AtJ1 is essential for normal plant growth, from embryogenesis to flowering and seed setting. Additionally, the ABA hypersensitivity of as793 suggests that AtJ1 may function as a negative regulator of ABA response. Keywords: ABA Abiotic stress Chaperone Flowering Hsp70 J protein. Abbreviations: ER, endoplasmic reticulum; Hsp70, heat shock protein 70; KO, knockout; MS. Murashige and Skoog; NEF, nucleotide exchange factor; RT–PCR, reverse transcription–PCR; TAIL-PCR, thermal asymmetric interlaced PCR.
Introduction Hsp70 family proteins (i.e. 70 kDa heat shock proteins; Hsp70s) are ubiquitous molecular chaperones that function in a variety of cellular processes (Kampinga and Craig 2010, Hartl et al. 2011). Under normal conditions, they facilitate the folding of nascent polypeptides, protein transport across cell membranes and oligomeric protein assembly. Under stress conditions, Hsp70s help refolding or degradation of stress-denatured proteins. The functions of Hsp70s depend on their binding and release of substrate proteins in an ATP-dependent cycle (Kampinga and Craig 2010, Hartl et al. 2011). However, Hsp70s do not
function alone. They require another class of chaperones, J protein/Hsp40 family members, and nucleotide exchange factors (NEFs) for their full activities. In the canonical mode of Hsp70 action, a J protein binds to a substrate protein and subsequently interacts with an ATP-bound Hsp70, delivering the substrate protein to the Hsp70–ATP complex. The binding of J protein and substrate stimulates the ATPase activity of Hsp70 and hydrolysis of the bound ATP, thereby causing conformational change and tighter binding of the substrate to the resulting ADP–Hsp70 complex. The J protein is then released from the complex, and ADP is exchanged with ATP by an NEF. Hsp70s have low affinity for substrates in their ATP-bound state. Thus, the nucleotide exchange is followed by substrate release, and a new cycle begins. In plants and other organisms, there are more J proteins than Hsp70s and NEFs (Kampinga and Craig 2010, Hartl et al. 2011). In Arabidopsis, for example, the Hsp70 family consists of 14 members, whereas the J protein family consists of at least 105 members (Lin et al. 2001, Sung et al. 2001, Finka et al. 2011). The functional diversity of the Hsp70 machinery is therefore determined, in major part, by J proteins. J proteins possess a highly conserved signature domain, which was originally found in the prototype J protein Escherichai coli DnaJ and thus named J domain (Kampinga and Craig 2010). The J domain consists of approximately 70 amino acids and contains an invariable His-Pro-Asp (HPD) tripeptide motif. The HPD motif is essential for the stimulation of ATPase activity of Hsp70s. Although they possess the highly conserved J domain, J proteins are diverse in their overall structure and grouped into three classes: Type I, II and III, or Type A, B and C. Type I J proteins have a domain structure similar to that of DnaJ, i.e. the N-terminal J domain is followed by a glycine/phenylalanine-rich region (G/F region), a zinc-finger motif (CXXCXGXG) and a C-terminal extension domain. Type II J proteins possess a J domain and a G/F region but lack the zinc-finger domain. Other J proteins belong to Type III. In Arabidopsis, Type III is the most abundant class (approximately 90 members) (Miernyk 2001, Rajan and D’Silva 2009, Finka et al. 2011, Chiu et al. 2013). Several different systems have been proposed for the nomenclature of Arabidopsis J proteins (Miernyk 2001, Rajan and D’Silva 2009, Finka et al. 2011). J proteins are localized not only in the cytosol but also in organelles such as the mitochondria, chloroplast, endoplasmic reticulum (ER) and nucleus (Miernyk 2001, Rajan and D’Silva
Plant Cell Physiol. 55(12): 2152–2163 (2014) doi:10.1093/pcp/pcu145, Advance Access publication on 13 October 2014, available online at www.pcp.oxfordjournals.org ! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
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Regular Paper
Departments of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, South Korea
Plant Cell Physiol. 55(12): 2152–2163 (2014) doi:10.1093/pcp/pcu145
Results Isolation of the ABA-hypersensitive mutant as793 We carried out an activation tagging screen to isolate ABA response mutants. A mutant library consisting of approximately 25,000 independent transgenic lines was prepared and screened for the isolation of mutants with altered ABA sensitivity, as described elsewhere (Park et al. 2011). A number of mutants were isolated, and one of them, designated as793 (ABA-hypersensitive 793), was characterized further in this study (Fig. 1A). Thermal asymmetric interlaced PCR (TAIL-PCR) (Liu et al. 1995) was carried out to determine the T-DNA insertion site in as793, and T-DNA was found to be located in one of the introns
of a gene (At1g28210) encoding a J protein, AtJ1 (Fig. 1B) (Kroczynska et al. 1996). Subsequent reverse transcription– PCR (RT–PCR) analysis to determine the AtJ1 transcript level indicated that its expression was completely abolished in the mutant (Fig. 1C). Thus, as793 is a knockout (KO) mutant of AtJ1. AtJ1 is also known as AtDjB1 (Miernyk 2001), atDiC23 (Rajan and D’Silva 2009) and DJC20 (Finka et al. 2011).
as793 is impaired in seedling growth and seed setting One of the most distinct phenotypes of as793 was poor seedling growth, i.e. the mutant seedlings grew very slowly compared with wild-type plants (Fig. 1D). To confirm that the phenotype is the loss-of-function phenotype of AtJ1, we acquired its KO mutant atj1 (SALK_049553), and, after the confirmation of T-DNA insertion in the annotated site, we analyzed its phenotypes in parallel with as793. AtJ1 expression is abolished completely in atj1 (Fig. 1C). Additionally, we prepared and analyzed AtJ1 complementation lines by transforming atj1 with a genomic DNA fragment containing the wild-type AtJ1 coding region and its promoter (Fig. 2). Like as793, atj1 seedlings also grew very slowly (Figs. 1D, 2). Consequently, both as793 and atj1 plants took longer than wild-type plants to reach the bolting stage and maturity (see below). In contrast, the growth of the complementation line was normal, indicating that the poor seedling growth is the KO phenotype of AtJ1. Another noticeable phenotype of as793 and atj1 was poor seed setting efficiency. As shown in Fig. 3A and B, the mutant plants had fewer siliques than wild-type plants and, frequently, silique formation was not complete. Close examination of the aberrant siliques revealed that they carried fewer seeds than wild-type siliques. Many aborted seeds, i.e. seeds which failed to develop, were observed, and, occasionally, partially developed seeds were found in the siliques (Fig. 3B). Thus, seed setting efficiency is impaired in the AtJ1 mutants.
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2009, Finka et al. 2011). As co-chaperones of Hsp70s, J proteins are involved in various cellular processes mentioned above. However, the specific functions of most J proteins in plants are poorly understood, except for a number of cases. An Arabidopsis Type I J protein GFA2/atDjA3, which is an ortholog of the yeast mitochondrial J protein MDJ1, is required for synergid cell death during megagametogenesis (Christensen et al. 2002). The cytosolic Type III J protein JAC1/atDjC18 is essential for phototropin-mediated chloroplast movement (Suetsugu et al. 2005). Five ER-localized J proteins, which are orthologs of yeast Sec63p, Scj1 or Jem1p, have been reported (Yamamoto et al. 2008). AtERdj2A/atDjC20, an ortholog of yeast Sec63p functioning in protein translocation across the ER membrane, is essential for seedling growth and pollen germination. AtERdj3A/atDjB9, AtERdj3B/atDjA8 and AtP58IPK/atDjC19, orthologs of yeast Scj1 and Jem1 functioning in protein folding in the ER, can partially complement the yeast jem1Dscj1 mutant. Cytoskeleton-associated Arabidopsis J proteins ARG1/atDjB11, ARL1/atDjB12 and ARL2/atDjB13 are highly homologous to each other and participate in the gravity signaling process (Sedbrook et al. 1999, Boonsirichai et al. 2003, Guan et al. 2003). The peroxisomal J protein GRV2/KAM2/ atDjC30 is involved in endosome formation and the tropic growth response (Silady et al. 2004, Tamura et al. 2007). Recently, J20/atDjC29 in plastids was shown to deliver the inactive deoxyxylulose 5-phosphate synthase enzyme to Hsp70 for proper folding or degradation (Pulido et al. 2013). In this study, we provide evidence that the Type III J protein AtJ1 is required for proper seedling growth and ABA response. In the course of our activation tagging screen to identify ABA response mutants, we isolated a mutant in which AtJ1 expression was abolished. Previously, Kroczynska et al. (1996) showed that recombinant AtJ1 stimulates the ATPase activity of both E. coli DnaK and maize endosperm Hsp70 in vitro, and the expression of the AtJ1 gene in an E. coli dnaJ deletion mutant can rescue the growth ability of E. coli under heat stress. Recently, Zhou et al. (2012) reported that AtJ1 facilitates thermotolerance by protecting cells against heatinduced oxidative damage. Here, we show that AtJ1 is essential for normal seedling growth, flowering time control and ABA response.
Flowering of as793 is delayed Although as793 and atj1 seedlings grew slowly and their seed setting efficiency was low, they grew to maturity, and mature atj1 and as793 plants were similar to wild-type plants in size and stature (Fig. 2D, E). However, it took longer for the mutant plants to reach the bolting stage (Fig. 3C). Under our growth conditions (i.e. 16 h light/8 h dark cycle), wild-type (Col-0) plants started bolting approximately 23 d after seed sowing, whereas the mutant plants bolted 32 d after sowing (Fig. 3D, E). Late bolting of as793 and atj1 plants is expected because the mutant seedlings grew slowly. However, it is also possible that flowering time might be delayed in the mutants. To address this possibility, we counted the number of rosette leaves at the bolting stage. Fig. 3D and E shows that the average number of rosette leaves of the wild-type plants and the complementation line was 13 at the bolting stage. In contrast, the number of rosette leaves of the mutant plants was 16 at the bolting stage. The result indicates that flowering time is delayed in the mutants. 2153
M. Y. Park and S. Y. Kim | AtJ1 is essential for normal plant growth
as793 is ABA-hypersensitive as793 was isolated based on its ABA hypersensitivity during early seedling growth (Fig. 1). To better understand the ABA sensitivity of as793, we investigated ABA-associated phenotypes at various developmental stages. First, we examined the ABA sensitivity during seed germination. Mature, dry seeds were plated on Murashige and Skoog (MS) medium containing various concentrations of ABA, and rates of germination (i.e. radicle emergence) were scored daily. In the medium lacking ABA, it took approximately 2 d for approximately 80% of the as793 and atj1 seeds to germinate, whereas nearly 100% of the wild-type and the complementation line seeds germinated within 1 d after imbibition (Fig. 4A). In the presence of increasing concentrations of ABA, germination rates of as793 and atj1 seeds decreased gradually, and their germination rates were approximately 30% at 1 mM ABA under our experimental conditions (Fig. 4B). Under the same conditions, germination of the wild-type and complementation line seeds was not noticeably affected, indicating that germination of the mutant seeds was more severely affected by ABA. We next examined the ABA sensitivity during the seedling establishment stage, by determining the effect of ABA on the 2154
cotyledon greening process (Fig. 4C, D). After imbibition, seeds were plated on MS medium containing various concentrations of ABA, and seedlings with green cotyledons were counted after prolonged time (i.e. 10 d) to allow full germination. Fig. 4C and D shows that wild-type and complementation line seeds germinated and all the seedlings developed to have green cotyledons at all concentrations of ABA tested. However, the cotyledon greening efficiency of as793 and atj1 seedlings decreased rapidly due to ABA, and few seedlings developed to have green cotyledons at ABA concentrations >0.5 mM. Similarly, we investigated the ABA effect on primary root elongation. Seeds were germinated on MS medium, and germinated seedlings were transferred to media containing various concentrations of ABA. Root growth was then measured 4 d after the transfer. Fig. 4E shows that root growth of the atj1 and as793 mutant plants was more severely inhibited by ABA than that of the wild-type plants at all doses of ABA. Root growth rates of the complementation line were similar to those of the wild-type plants. Thus, AtJ1 KO mutants were ABA-hypersensitive during seed germination and post-germination growth.
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Fig. 1 Isolation of the ABA-hypersensitive mutant as793. (A) ABA sensitivity of activation-tagged mutants. Seeds from putative ABA response mutants including as793 were germinated and grown for 6 d in MS medium (MS) or MS medium with ABA (0.5 mM). (B) T-DNA insertion sites in as793 and atj1 (SALK_049553) are shown schematically. LB indicates the border sequence. Filled and open boxes indicate the exons and untranslated regions, respectively. (C) AtJ1 transcript levels were determined by semi-quantitative RT–PCR. Actin-1 was used as a reference gene. (D) Mutant seedlings grown for 10 d are shown.
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Fig. 2 AtJ1 complementation lines and growth of the atj1 and as793 mutant. (A) Growth of the wild type (Col-0), atj1 and complementation lines (atj1/AtJ1). Plants were grown in MS medium for 10 d. (B) Expression levels of AtJ1 relative to the wild-type level in the complementation lines are shown. (C) Growth of wild-type, atj1, as793 and atj1/AtJ1 (#10) plants. Plants were grown in soil for 3 (top), 5 (middle) or 6 (bottom) weeks in soil. (D, E) Fresh weight of shoot (C) or root (D) from plants grown for the indicated times. Bars represent the SD (n = 15 each).
The hypersensitivity of as793 is ABA-specific Our results indicated that germination and seedling growth of as793 and atj1 were hypersensitive to ABA. To address whether the altered hormone response was specific to ABA or not, we examined the effects of other hormones on the growth of the mutant plants. Seeds were germinated and grown in media containing various hormones or hormone precursors, such as ABA, 1-aminocyclopropane-1-caboxylic acid (ACC), benzyladenine (BA), brassinolide (BL), GA3 or IAA. Under our experimental condition, GA3 and auxin had little effect on seedling growth, whereas ACC, BA and BL had inhibitory effects. However, these hormones affected mutant seedlings and wild-type plants to similar degrees, and the hypersensitivity was observed only with ABA (Supplementary Fig. S1). The result suggests that hypersensitive response is ABA-specific.
as793 is hypersensitive to glucose and high osmolarity during early seedling growth The endogenous ABA level is increased by glucose, and ABA plays a positive role in glucose-mediated inhibition of seedling establishment, i.e. cotyledon expansion and greening (Leon and
Sheen 2003). To investigate whether AtJ1 is involved in the process, we examined the glucose effect on the cotyledon greening process. Fig. 5A and B shows that growth of the mutants was more severely inhibited by glucose than that of wildtype plants. For example, shoot growth of the mutants was almost completely inhibited at 3% glucose. On the other hand, wild-type and complementation line plants were not affected by the low concentration of glucose, and approximately 70% of their seedlings still turned green at 5% glucose. It has been well established that ABA mediates the salt response (Xiong and Zhu 2002). To determine whether AtJ1 contributes to the salt response, we examined the salt effect on germination and post-germination growth of as793. As shown in Fig. 5C, salt inhibition of germination was more severe in the atj1 and as793 mutant plants than in the wild-type plants. For example, germination of the wild-type and complementation line seeds was not affected noticeably up to 125 mM NaCl under our experimental conditions, but germination rates of the mutant seeds were reduced from 93% to approximately 25%. Similarly, the cotyledon greening process of the mutant seedlings was more severely inhibited by salt than that of wildtype and complementation line plants (Fig. 5D, E). We also 2155
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investigated the effect of high osmolarity on seed germination and cotyledon greening and observed a hypersensitive response during both germination and the cotyledon greening process (Supplementary Fig. S2). Collectively, these results indicate that as793 and atj1 mutants are hypersensitive to salt and high osmolarity during the early seedling growth stage.
as793 is drought-tolerant Our results indicated that AtJ1 is required for the normal ABA response. Because ABA controls response to abiotic stresses such as drought and high salinity (Xiong et al. 2002, Finkelstein 2013), we investigated whether drought tolerance was affected in as793 and atj1. Seedlings of similar developmental stages, i.e. 2-week-old wild-type or complementation line plants and 3-week-old as793 and atj1 plants, were subjected 2156
to water deficit conditions by withholding water for 14 d. The plants were re-watered at the end of the treatment, and survival rates were scored 3 d after re-watering. Fig. 6 shows that survival rates of as793 and atj1 plants were higher than those of the wild-type and complementation line plants: The survival rates of as793 and atj1 plants were approximately 80%, whereas the survival rates of wild-type and complementation line (atj1/ AtJ1) plants were approximately 30%. The result indicates that AtJ1 mutants are drought-tolerant, and thus suggests that AtJ1 is a negative regulator of the water-deficit response. To address the possible mechanism of the enhanced drought response, we determined the transpiration rates using detached leaves. Fig. 6C shows that the water loss rates of mutant leaves were lower than those of the wild-type or complementation line leaves, indicating that lower
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Fig. 3 Silique formation and flowering of AtJ1 mutants. (A) Siliques of 47-day-old plants. (B) Seed development in wild-type, atj1 and as793 siliques. White arrows, undeveloped ovules; white stars, aborted seeds. (C) Wild-type (Col-0), atj1, as793 and atj1/AtJ1 (#10) plants grown for 4 (top panel) or 5 (bottom panel) weeks. (D, E) Bolting time and the number of leaves in wild-type (Col-0), atj1, as793 and atj1/AtJ1 (#10) plants at the bolting stage. Plants were grown in long-day conditions, and the numbers on the y-axis represent days or leaf numbers. The data represent mean values and the small bars indicate the SE (n = 15).
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Fig. 4 ABA sensitivity of AtJ1 mutants. (A) Germination of AtJ1 mutants. Germination rates of wild-type and mutant seeds were scored in the medium lacking ABA. Experiments were carried out in triplicate (n = 50 each). (B) ABA dose–response of germination. Germination of the wild type, atj1, as793 and the complementation line (atj1/AtJ1 #10) on medium containing various concentrations of ABA was scored 3 d after imbibition. Experiments were carried out in triplicate (n = 50 each), and the small bars indicate the SE. (C) ABA dose–response of shoot development of the wild type, atj1, as793 and atj1/AtJ1 (#10). Seeds were germinated and grown for 10 d in media containing various concentrations of ABA before photographs were taken. (D) Cotyledon greening efficiency of the seedlings in (B). Experiments were carried out in triplicate (n = 50 each), and the small bars indicate the SE. (E) Root elongation assay. Seedling were germinated and grown in a medium lacking ABA for 3 d, and the seedlings were transferred to a medium containing various concentrations of ABA. Root elongation was measured 4 d after the transfer. Experiments were carried out in triplicate (n = 6 each), and the small bars indicate the SE.
transpiration rates of the mutant plants might have contributed to the enhanced drought tolerance of as793 and atj1. As mentioned earlier, the ABA level increases under water-deficit conditions, and several osmolytes, especially proline (Kishor
1995), are known to accumulate to enhance osmotolerance. Therefore, we determined their contents in as793 and found that, although ABA contents did not change noticeably, proline contents were higher in as793 than in wild-type plants 2157
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(Supplementary Fig. S3). Consistent with this observation, the expression level of the proline biosynthetic gene P5CS1 (Yoshiba et al. 1995) under normal conditions was higher (i.e. approximately three times) in as793 compared with the wildtype level (Supplementary Fig. S3). Under high salt conditions, the P5CS1 level in as793 was similar to the wild-type level. We also observed an increase in the expression level of the proline catabolic gene ProDH (Kiyosue et al. 1996) under high salt 2158
conditions (Supplementary Fig. S3). Thus, our results indicate that AtJ1 affects the expression of proline metabolic genes.
The expression of genes involved in embryogenesis, flowering time control and stress response is altered in as793 To address the possible mode of AtJ1 function, we examined whether the expression of various genes associated with the
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Fig. 5 Glucose and salt sensitivities of AtJ1 mutants. (A) Glucose dose–response of shoot development. Seeds of the wild type (Col-0), atj1, as793 and atj1/AtJ1 (#10) were germinated and grown for 10 d in media containing various concentrations of glucose. (B) Cotyledon greening efficiency of the seedlings in (A). Experiments were carried out in triplicate (n = 50 each), and the small bars indicate the SE. (C) Salt dose–response of germination. Seed germination was scored 3 d after imbibition on media containing various concentrations of salt (NaCl). Experiments were carried out in triplicate (n = 50 each), and the small bars indicate the SE. (D) Salt dose–response of shoot development. Seeds were germinated and grown for 10 d in media containing various concentrations of salt (NaCl). (E) Cotyledon greening efficiency of the seedlings in (C). Experiments were carried out in triplicate (n = 50 each), and the small bars indicate the SE.
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Fig. 6 Drought tolerance of AtJ1 mutants. (A) Water was withheld from plants of similar developmental stages (top panel) for 14 d and then the plants were watered. The picture shows the plant 3 d after the re-watering. (B) Survival rates. The values represent the mean of three independent experiments (n = 20 each), and the small bars represent the SE. (C) Transpiration rates. Leaves were detached from plants of a similar developmental stage (i.e. 4th and 5th true leaves from 21-day-old Col-0 or atj1/AtJ1 (#10) plants and 31-day-old as793 or atj1 plants) and leaf weight was measured at the indicated time interval. The values indicate weight relative to initial weight. Experiments were carried out in triplicate (n = 10 each).
phenotypes described above was altered. First, we compared the expression levels of a number of embryogenesis-related genes in the mutants with their wild-type levels by carrying out real-time RT–PCR. Fig. 7A shows that the expression of a number of genes playing important regulatory roles during embryogenesis (Capron et al. 2009, De Smet et al. 2010) was altered. WOX2, which regulates apical pattering during the early embryogenesis stage, was up-regulated approximately 3fold in as793 and atj1. On the other hand, several other genes, such as PIN1, MP/ARF5, BSK12/SSP and STM, were significantly down-regulated. Next, we determined the expression levels of flowering time control genes. As shown in Fig. 7B, the transcript level of the floral repressor FLC in the mutants was approximately three times the wild-type level. In contrast, expression levels of several positive regulators, such as FT, LFY and SOC1, were much lower (10–20%) than their wild-type levels. We also determined the expression levels of ABA- and stress-regulated genes (Fig. 7C). Among the known ABA signaling components, the expression level of ABI5, which is one of the key positive regulators of ABA response (Finkelstein 2013), was approximately 2.5 times the wild-type level in as793 and atj1. Our result further showed that a number of
stress-regulated, in particular, cold-regulated, genes were down-regulated in the mutants (Fig. 7C).
Discussion J proteins function as co-chaperones of Hsp70s by activating the ATPase activity of Hsp70s and/or by delivering substrates to Hsp70s. As mentioned in the Introduction, a number of Arabidopsis J proteins have been reported, and these studies have shown that they are involved in various cellular processes, such as gravity and tropic responses, light-dependent chloroplast movement, gametogenesis, pollen germination, protein translocation and maintenance of proper enzyme function. Considering the large number of Arabidopsis J proteins, however, only a fraction of them have been reported so far, and the specific functions of most J proteins remain to be determined. AtJ1 is a Type III Arabidopsis J protein. The gene encoding AtJ1 is constitutively expressed in various tissues and is heatinducible (Zhou et al. 2012). Previously, Kroczynska et al. (1996) showed that recombinant AtJ1 can stimulate the ATPase activity of HsP70s and is imported into mitochondria. Zhou et al. 2159
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Fig. 7 Transcript level changes in AtJ1 mutants. (A) Transcript levels of embryogenesis-related genes relative to the wild-type levels are shown: WOX2 (At5g59340), PIN1 (At1g73590), ARF5 (At1g19850), BSK12/SSP (At2g17090) and STM (At1g62360). (B) Relative expression levels of flowering time control genes: FLC (At5g10140), FT (At1g65480), LFY (At5g61850) and SOC1 (At2g45660). (C) Relative induction levels of ABA/stress-responsive genes: ABI5 (At2g36270), CBF1 (At4g25490), CBF2 (At4g25470), CBF3 (At4g25480), COR15A (At2g42540) and RAB18 (At5g66400). For (A) and (B), RNA was isolated from plants grown under normal conditions. For (C), RNA was isolated from plants subjected to low temperature (0 C) for 3 h, except for ABI5 and their relative induction levels compared with untreated levels are shown. Real-time PCRs were done in duplicate, and the small bars indicate the SE.
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Further expression analyses showed that the expression of additional genes involved in plant development and stress response was altered in as793 (Supplementary Figs. S3–S5). These include a number of genes regulating embryogenesis (ABCB1, PID, SHR, etc.) (Supplementary Fig. S5A) and seed dormancy/germination (SOMNUS and GA3ox1) (Supplementary Fig. S5B). Changes in the expression levels of stress-responsive genes were also detected (Fig. 7C; Supplementary Figs. S3, S4). In particular, RD20 expression was up-regulated 3- to 5-fold in as 793 (Supplementary Fig. S4). RD20 expression is induced by ABA and abiotic stresses, and it has been demonstrated to mediate ABA response and drought tolerance (Aubert et al. 2010, Blee et al. 2014). Thus, AtJ1 affects the expression of a large number of genes that regulate plant development and stress response. In summary, our results show that, in addition to its function in mitochondria, AtJ1 is involved in other cellular processes that affect plant development, ABA response and stress tolerance. At present, the putative substrate proteins of AtJ1, which are responsible for these cellular activities, are not known, and the mechanism of AtJ1 function remains to be determined. However, our data strongly suggest that the function of AtJ1 entails the modulation of gene expression.
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(2012) recently reported that AtJ1 functions with a mitochondrial Hsp70 to protect cells against heat-induced oxidative damage by maintaining cellular redox homeostasis. Our results further indicate that AtJ1 is essential for normal plant growth, i.e. its mutants are defective in various aspects of plant growth: embryogenesis, seed germination, seedling growth, transition to flowering and seed setting. AtJ1 KO mutants, as793 and atj1, grew very poorly, especially during the early stage of seedling growth (Figs. 1, 2). Both root and shoot growth of seedlings was slow and, as a consequence, it took much longer (approximately 9 d) for the mutant seedlings to reach the bolting stage (Fig. 3). However, plant morphology was normal and mature mutant plants were of the same size as the wild-type plants (Fig. 2), indicating that the developmental process itself was not affected in the mutants. In addition to slow seedling growth, transition to the flowering stage was also delayed: mutant plants started to bolt at the 16 leaf stage, whereas wild-type plants bolted at the 13 leaf stage. The AtJ1 mutant plants also exhibited poor seed setting efficiency. Embryo development was frequently aborted at the initial stage, silique formation was not complete and fewer siliques were observed. Furthermore, the mature embryos germinated slowly (Fig. 4A), and greater seed dormancy was observed (data not shown). In addition to general plant growth/development, the ABA response was altered in the AtJ1 mutants. The mutants were hypersensitive to ABA inhibition during germination and post-germination growth. Additionally, the mutant seedlings were hypersensitive to glucose, whose effect is mediated by ABA. Consistent with the ABA hypersensitivity, the transpiration rates of the the mutant seedlings were lower, and the mutant plants were drought-tolerant (Fig. 6). We also investigated tolerance towards other stresses and found that seedling growth was hypersensitive to high salt (Fig. 5) and high osmolarity (Supplementary Fig. S2) during seedling establishment (i.e. germination and the cotyledon greening stage). Others reported that AtJ1 is a mitochondrial protein (Kroczynska et al. 1996, Zhou et al. 2012). Thus, it can be speculated that the poor plant growth and altered ABA response may be due to the defect in the function of mitochondria, which provides the energy required for cellular activities. However, our expression analyses (Fig. 7) demonstrate that AtJ1 affects the expression of a large number of genes involved in various developmental processes. Some of the genes are regulatory genes. For instance, we observed significant downregulation of a number of genes that play important roles in embryogenesis, such as PIN1, ARF5, SSP and STM (Fig. 7A). Moreover, we observed altered expression of genes controlling flowering time. FLC, which is a repressor of flowering, was enhanced, whereas positive regulators, such as FT, LFY and SOC1, were down-regulated (Fig. 7B). This altered gene expression pattern is consistent with the late flowering phenotype of the AtJ1 mutants. Also, it is noteworthy that the expression of ABI5, which is a key ABA signaling component regulating seed dormancy, germination and early seedling growth (Finkelstein 2013), was up-regulated in as793 (Fig. 7C).
Materials and Methods Isolation of the as793 mutant The generation of activation-tagged transgenic plants and the screen for ABAhypersensitive mutants has been described elsewhere (Park et al. 2011). The TDNA insertion site in the as793 mutant was determined by TAIL-PCR (Liu et al. 1995). The T-DNA insertion site was then confirmed by sequencing the DNA fragment amplified using the T-DNA border sequence 50 -AAT AAC GCT GCG GAC ATC TA-30 and the gene-specific primer 50 -TCC ATG ATC TAC AAA AAA GCC TTG C-30 . RT–PCR analysis was performed using the primers 50 -CAA CTT TCA CTA GGG ATG GCT CA-30 and 50 -CGG AAG CGA ACA TAC TGA TCT C-30 .
Analysis of AtJ1 knockout lines The KO line (SALK_049553) of AtJ1, atj1, was obtained from the Arabidopsis Stock Center. The knockout plants were kanamycin susceptible. To purify homozygous lines, PCR was performed with genomic DNA isolated from next-generation plants grown individually. For the genotyping analysis, the primer set 50 -CAA CTT TCA CTA GGG ATG GCT CA-30 (forward), 50 -CGG AAG CGA ACA TAC TGA TCT C-30 (reverse) and 50 -CAG AAA TGG ATA AAT AGC CTT GCT T-30 (left border) was used. For semi-quantitative RT-PCR in Fig, 1, 50 -CAA CTT TCA CTA GGG ATG GCT CA-30 and 50 -CGG AAG CGA ACA TAC TGA TCT C-30 were used.
Generation of complementation lines and phenotype analysis To prepare complementation lines, a genomic DNA fragment containing the entire coding region of AtJ1 was amplified using the primer set 50 -ATG CGA AGA TTC AAC TGG GTT C-30 and 50 -cga gct cCA GCT TAA CTT GTT TGA TGT C-30 , and, after SacI digestion, cloned into AtJ1 promoter-pBI101.2. AtJ1 promoter-pBI101.2, in turn, was prepared by cloning the 1.7 kb promoter fragment amplified using the primer set 50 -gcg tcg acA GTG TCT TTA TCA TTT CGG ACA-30 and 50 -cgg gat ccG ACT CTT GTG AAT TCA AAG CTA AAC-30 into pBI101.2 and then removing the b-glucuronidase (GUS) coding region after SmaI/SacI digestion. The construct was then introduced into Agrobacterium tumefaciens strain GV3101, and the atj1 mutant
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RNA isolation and expression analysis Seedling RNA was isolated employing the Qiagen RNeasy plant mini kit. Seed RNA isolation was carried out employing the Sigma SpectrumTM plant total RNA kit (Sigma). For RT–PCR analysis, possible contaminating DNA was removed from RNA samples by DNase I treatment. The first-strand cDNA was synthesized using Superscript III (Invitrogen) according to the supplier’s instructions. For semi-quantitative RT–PCR, cDNA amplification was carried out within a linear range using gene-specific primers. For real-time RT–PCR, the cDNA amplification was performed with SsoFast EvaGreen supermix in a BioRad CFX96 Real-Time PCR Systems (Bio-Rad), and quantitation was carried out using the CFX96 Real-Time PCR System’s software. Actin-1 was employed as a reference gene. The primers used in the expression analysis of various genes are shown in Supplementary Tables S1 and S2.
Supplementary data Supplementary data are available at PCP online.
Funding This work was supported the Rural Development Administration, Republic of Korea [the Next Generation BioGreen 21 Program (grant PJ00819804 to S.Y.K.)]; the Ministry of Education, Science and Technology (MEST) [the Mid-career Researcher Program through an NRF grant [No. 2011-0015455 to S.Y.K.].
Acknowledgments The authors are grateful to the Kumho Life Science Laboratory of Chonnam National University for providing equipment and plant growth facilities. We thank Seul-bi Lee and Sun-geum Jeung for their technical assistance.
Disclosures The authors have no conflicts of interest to declare.
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