aba-dependent and aba-independent functions of rcar5/pyl11 ...€¦ · 8/2/2019 · 12....

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ABA-dependent and ABA-independent functions of RCAR5/PYL11 in response 1

to cold stress 2

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Chae Woo Lim and Sung Chul Lee* 5

Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea 6

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*Corresponding author 8

Professor S. C. Lee 9

84 Heukseok-Ro, Dongjak-Gu, Department of Life Science, Chung-Ang University, 10

Seoul 06974, Korea 11

E-mail: [email protected] 12

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CW Lim: [email protected] 14

SC Lee: [email protected] 15

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not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 2, 2019. ; https://doi.org/10.1101/723627doi: bioRxiv preprint

https://doi.org/10.1101/723627

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Abstract 1

Arabidopsis thaliana has 14 abscisic acid (ABA) receptors—PYR1/PYLs/RCARs—which 2

have diverse and redundant functions in ABA signaling; however, the precise role of these 3

ABA receptors remains to be elucidated. Here, we report the functional characterization of 4

RCAR5/PYL11 in response to cold stress. Expression of RCAR5 gene in dry seeds and 5

leaves was ABA-dependent and ABA-independent, respectively. Under cold stress 6

conditions, seed germination was markedly delayed in RCAR5-overexpressing 7

(Pro35S:RCAR5) plants, but not in Pro35S:RCAR5 in ABA-deficient (aba1-6) mutant 8

background. Leaves of Pro35S:RCAR5 plants showed enhanced stomatal closure—9

independent of ABA—and high expression levels of cold, dehydration, and/or ABA-10

responsive genes; these traits conferred enhanced freezing tolerance. Our data suggest that 11

RCAR5 functions in response to cold stress by delaying seed germination and inducing rapid 12

stomatal closure via ABA-dependent and ABA-independent pathways, respectively. 13

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Keywords: abscisic acid, cold stress, RCAR, seed germination, stomatal closure 15


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Introduction 1

The plant hormone abscisic acid (ABA) plays a key role in growth and development, as well 2

as in adaptive mechanisms to unfavorable conditions such as regulation of seed dormancy, 3

germination, and stomatal opening and closure (Cutler et al., 2010, Hubbard et al., 2010). 4

During seed maturation, ABA accumulates in seeds, inducing and maintaining seed 5

dormancy and inhibiting seed germination by preventing water uptake and endosperm 6

rupture (Müller et al., 2006, Seo et al., 2006). Seed germination leads to a rapid decline in 7

ABA content and suppression of ABA signaling, suggesting that ABA is an important 8

hormone inhibiting seed germination until favorable growth conditions prevail (Finkelstein et 9

al., 2008, Shu et al., 2016). In vegetative tissue, drought stress induces ABA accumulation, 10

leading to stomatal closure and induction of stress-related genes (Cutler et al., 2010). In 11

Arabidopsis, ABA levels increase transiently and less markedly (2-fold) in response to cold 12

stress than in response to drought stress (approx. 20-fold) (Lång et al., 1994). Several 13

studies have shown that ABA is involved in cold stress responses; these studies have 14

examined cold stress-induced ABA biosynthesis (Cuevas et al., 2008, Lång et al., 1994); 15

absence of cold acclimation in ABA-deficient mutants (Gilmour & Thomashow, 1991, Xiong 16

et al., 2001); and ABA induction of cold-responsive genes, mainly CBF genes (Knight et al., 17

2004, Lee & Seo, 2015). However, in comparison with the well documented role of ABA in 18

drought stress, the function of this plant hormone in cold stress and acclimation remains 19

controversial. 20

ABA is perceived by the PYRABACTIN RESISTANCE/PYRABACTIN RESISTANCE-21

LIKE/REGULATORY COMPONENT OF ABA RECEPTOR (PYR/PYL/RCAR; hereafter 22

referred to as RCARs) protein family, which consists of 14 members in Arabidopsis. RCARs 23

belong to the START-domain superfamily and are divided into three subfamily groups 24

according to sequence homology (Ma et al., 2009, Park et al., 2009). In response to 25


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environmental stresses, ABA is rapidly synthesized and binds to RCARs (Cutler et al., 2010). 1

ABA-bound RCARs selectively interact with and inhibit clade A protein phosphatase 2Cs 2

(PP2Cs), including ABA-insensitive 1 (ABI1), ABI2, hypersensitive to ABA1 (HAB1), HAB2, 3

ABA-hypersensitive germination 1 (AHG1), AHG3/PP2CA, highly ABA-induced PP2C gene 1 4

(HAI1), HAI2, and HAI3 (Bhaskara et al., 2012, Gonzalez-Guzman et al., 2012, Ma et al., 5

2009, Nishimura et al., 2010, Park et al., 2009). Among potential RCAR–PP2C interactions, 6

113 pairings are known to be functional (Tischer et al., 2017). As a consequence of these 7

interactions, PP2C inhibition of sucrose non-fermenting 1-related protein kinase 2s (SnRK2s) 8

is cancelled, resulting in phosphorylation and activation of downstream components, such as 9

transcription factors and ion channels (Furihata et al., 2006, Geiger et al., 2009, Lee et al., 10

2009). In this process, bZIP transcription factor ABI5—the core ABA signaling component—11

is phosphorylated by ABA-activated SnRK2.2, SnRK2.3, and SnRK2.6 (Nakashima et al., 12

2009) and subsequently regulates expression of stress adaptation genes, e.g., late 13

embryonic and abundant genes such as EARLY METHIONINE-LABELED 1 (EM1) and EM6 14

(Finkelstein & Lynch, 2000). 15

RCARs have diverse and redundant functions in ABA and drought-stress signaling 16

(Antoni et al., 2013, Hao et al., 2011, Zhao et al., 2016, Zhao et al., 2013). With the 17

exception of RCAR7, RCARs induce expression of ABA-responsive genes with highly 18

variable expression levels. Also, RCAR levels—with the exception of RCAR4—significantly 19

suppressed by ABA deficiency (Fujii et al., 2009, Ma et al., 2009, Szostkiewicz et al., 2010, 20

Tischer et al., 2017, Zhao et al., 2013). This may be explained by differing ABA affinities and 21

ABA dependencies of RCARs and/or variations in abundance of RCARs and their target 22

PP2Cs (Tischer et al., 2017). ABA affinities of RCARs are influenced by oligomerization 23

states; monomeric RCARs (RCAR1, RCAR3, RCAR4, RCAR8, RCAR9, and RCAR10) have 24

higher affinities than dimeric RCARs (RCAR11, RCAR12, RACR13, and RCAR14) (Dupeux 25


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et al., 2011, Hao et al., 2011). Moreover, monomeric RCARs interact with PP2Cs mainly in 1

an ABA-independent manner, whereas dimeric RCARs bind to PP2Cs in an ABA-dependent 2

manner (Hao et al., 2011, Tischer et al., 2017). In most RCARs, functional redundancy 3

interferes with analysis of biological function in ABA signaling based on single-gene 4

mutations (Park et al., 2009). In contrast, mutation in multiple RCARs negatively influences 5

ABA sensitivity and drought resistance, whereas overexpression of RCARs positively 6

regulates ABA signaling and drought response (Gonzalez-Guzman et al., 2012, Lim et al., 7

2014, Santiago et al., 2009, Zhao et al., 2014). Loss-of-function of RCAR3/PYL8 resulted in 8

reduced sensitivity to ABA-induced inhibition of primary root growth (Antoni et al., 2013). In 9

combination with RCAR2/PYL9 and RCAR3/PYL8 functions in regulating of lateral root 10

growth via interaction with MYB transcription factor MYB77 and MYB44 by independent 11

manner of the core ABA signaling pathway (Xing et al., 2016, Zhao et al., 2014). 12

RCAR2/PYL9 overexpression conferred drought resistance by promoting ABA-mediated leaf 13

senescence (Zhao et al., 2016). This suggests that RCARs have distinct functions, which are 14

associated with different tissues, developmental stages, and specific environmental 15

conditions (Sun et al., 2017). However, non-redundant functionality of RCAR in an ABA-16

dependent and/or ABA-independent manner remains to be elucidated. Here, we attempted 17

to functionally characterize RCAR5 in response to cold stress. 18

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Results 20

RCARs are differentially regulated in ABA-deficient seeds 21

Several studies based on public microarray data have shown that expression patterns of 22

RCARs vary among different tissues and in response to ABA and abiotic stresses, 23

suggesting substantial functional differences among these genes (Gonzalez-Guzman et al., 24

2012, Kilian et al., 2007, Klepikova et al., 2016, Park et al., 2009, Santiago et al., 2009, 25


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Szostkiewicz et al., 2010, Winter et al., 2007). However, RCAR gene expression patterns in 1

seeds remain unclear. We investigated expression levels of RCARs in dry seeds of 2

Arabidopsis thaliana Columbia-0 (Col-0) and Landsberg erecta (Ler) ecotypes (Appendix Fig 3

S1A). Transcripts of six RCARs—RCAR1, RCAR2, RCAR3, RCAR5, RCAR6, and 4

RCAR11—were relatively abundant; further, RCAR1, RCAR2, RCAR5, and RCAR6 were 5

significantly downregulated after imbibition (Appendix Fig S1B and C). ABA is a key hormone 6

involved in the induction and maintenance of seed dormancy, and its level decreases rapidly 7

following imbibition (Kushiro et al., 2004, Millar et al., 2006). Hence, we postulated that 8

transcriptional alteration of RCAR1, RCAR2, RCAR5, and RCAR6 is associated with 9

endogenous ABA level in seeds. We examined expression levels of RCARs in the ABA-10

deficient mutants aba1-6 (Col-0 background) and aba1-3 (Ler background). In comparison 11

with wild-type (WT) seeds, ABA-deficient seeds showed upregulated expression of RCAR9 12

and RCAR10, but downregulated expression of RCAR1, RCAR2, RCAR5, and RCAR6 (Fig 13

1A). Hence, RCAR expression in seeds may be influenced by endogenous ABA level, and 14

downregulation of RCAR1, RCAR2, RCAR5, and RCAR6 may be associated with breaking 15

of ABA-induced seed dormancy. 16

Dormancy is an important adaptive trait that improves plant survival by inhibiting seed 17

germination under unfavorable conditions, including cold stress (Finkelstein et al., 2008). In 18

general, cold stratification (2–5°C for 2–4 days) causes a release of seed dormancy in 19

Arabidopsis and promotes seed germination (Baskin & Baskin, 2004). We sowed freshly 20

harvested seeds of Arabidopsis Col-0 on 0.5 MS agar plates, stratified the seeds at 4 ± 1°C 21

in the dark for 2 days, and subjected the stratified seeds to cold stress by continuous 22

incubation at 4 ± 1°C. Under cold stress conditions, germination was delayed; seeds started 23

to germinate 10–11 days after incubation (DAI) and all seeds had germinated 16 DAI (Fig 24

1B). We investigated the functional role of RCARs in sensitivity of imbibed seeds to cold 25


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stress. First, we examined the expression patterns of RCARs during seed germination under 1

cold stress conditions (Fig 1C). Genes involved in ABA response and GA-mediated cell wall 2

modification are differentially expressed during seed germination (Liu et al., 2016). We 3

amplified the ABA-responsive gene ABI5 and the cell wall-related gene EXPANSIN3 (EXP3) 4

as a control; at 8 DAI, transcripts of ABI5 and EXP3 were significantly downregulated and 5

upregulated, respectively. Consistent with data obtained using Arabidopsis thaliana Col-0 6

and Ler ecotypes (Appendix Fig S1B and C), expression levels of RCAR1, RCAR2, RCAR5, 7

and RCAR6 decreased significantly during seed germination; however, we determined no 8

significant change in expression levels of RCAR3 and RCAR11 (Fig 1C). 9

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RCAR5 plays a key role in delay of seed germination under cold stress conditions 11

Several studies have shown that ABA signaling is related to the cold stress response (Ding 12

et al., 2015, Knight et al., 2004, Kreps et al., 2002, Lee et al., 2010). To analyze the 13

functional role of RCARs in seed germination under cold stress conditions, we generated 14

Arabidopsis transgenic plants overexpressing each RCAR under the control of the 35S 15

promoter. In comparison with WT plants, the 14 transgenic lines showed high expression 16

levels of each RCAR gene (>30-fold to 300-fold) (Appendix Fig S1D) and also high 17

sensitivity to ABA during seed germination and seedling growth (Fig 2A). To examine the 18

mechanism by which enhanced ABA sensitivity influences seed germination or seedling 19

growth of transgenic plants under cold stress conditions, freshly harvested seeds of each 20

line were sown on 0.5 MS agar plates and stratified at 4 ± 1°C in the dark for 2 days. Under 21

normal growth conditions (24°C), germination rates did not differ significantly between 22

transgenic plants and WT plants (Fig 2A and Appendix Fig S1E–G). However, under cold 23

stress conditions, the germination rates of transgenic lines Pro35S:RCAR2 and 24

Pro35S:RCAR5 at 14 DAI were significantly lower (56.0% and 25.9%, respectively) than 25


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those of WT plants (64.4%) (Fig 2B). At 21 DAI, the number of etiolated seedlings was 1

significantly lower in Pro35S:RCAR5 lines than in WT and Pro35S:RCAR2 lines but did not 2

differ significantly between WT and Pro35S:RCAR2 lines. We further observed phenotypic 3

differences in seedling growth at 8°C; at this temperature, germination of WT and transgenic 4

lines was enhanced; however, the germination rate was significantly lower in Pro35S:RCAR5 5

lines than in WT and the remaining 13 transgenic lines (Fig 2A). To determine whether there 6

was a loss of seed viability in the Pro35S:RCAR5 line, seeds of Pro35S:RCAR5 (#1) and 7

Pro35S:RCAR5 (#2) were incubated at 4 ± 1°C for 18 days and maintained at 24°C. After 4 8

days, all seeds germinated and developed into seedlings (Fig 2C). 9

To examine delayed germination of Pro35S:RCAR5 seeds at the molecular level, we 10

performed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. 11

We examined expression levels of ABA-responsive genes in dry seeds of Pro35S:RCAR5 12

and WT lines (Fig 2D). The ABA-responsive genes ABA INSENSITIVE 3 (ABI3), ABI4, ABI5, 13

MOTHER OF FT AND TFL1 (MFT), TANDEM CCCH ZINC FINGER PROTEIN 5 (TZF5), 14

EARLY METHIONINE-LABELED 1 (EM1), and EM6 were more strongly expressed in 15

Pro35S:RCAR5 seeds than in WT seeds. However, we determined no significant difference 16

in expression level of the XERICO gene—which promotes ABA accumulation in seeds 17

(Zentella et al., 2007)—between Pro35S:RCAR5 and WT seeds. During germination at 4°C, 18

expression levels of ABA-responsive genes were significantly higher in Pro35S:RCAR5 19

seeds than in WT seeds; however, expression of cell wall-related genes—EXPANSIN3 20

(EXP3), EXP9, XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH5), and 21

XTH31—was downregulated in Pro35S:RCAR5 seeds (Fig 2E). Our data suggest that 22

RCAR5 functions in ABA-mediated inhibition of seed germination under cold stress 23

conditions. 24

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Cold stress-induced germination delay of Pro35S:RCAR5 seeds occurs via an ABA-1

dependent pathway 2

Pro35S:RCAR5 seeds showed high expression levels of ABA-responsive genes; hence, we 3

postulated that RCAR5 overexpression delays seed germination via an ABA-dependent 4

pathway. WT and Pro35S:RCAR5 seeds were placed on 0.5 MS medium supplemented 5

with norflurazon (NF)—an ABA biosynthesis inhibitor—and germinated at 4°C in the dark. 6

Supplementation with NF increased the germination rate in a dose-dependent manner; 7

notably, at 14 DAI, the germination rate of Pro35S:RCAR5 seeds was 2-6 fold higher in the 8

presence than in the absence of NF (Fig 3A and B). This enhanced germination was 9

observed at all the investigated time points. In contrast, at 16 DAI, we observed no influence 10

of NF on germination of WT seeds. In contrast to NF, ABA strongly inhibited seed 11

germination and caused a 4-day delay in germination of WT seeds (Fig 3C). At 18 DAI, the 12

germination rates of WT and Pro35S:RCAR5 seeds on 0.5 MS medium supplemented with 13

0.75 μM ABA were 77.1% and <3%, respectively. As predicted, NF was antagonistic to ABA-14

mediated inhibition of seed germination. In the presence of NF, the germination rate of WT 15

seeds at 14 DAI was 48.2–64.5% (vs. 5.6% in the absence of NF); moreover, in the 16

presence of ABA, NF enhanced germination of Pro35S:RCAR5 seeds by up to 26%. To 17

verify germination delay of Pro35S:RCAR5 seeds using genetic analysis, we overexpressed 18

RCAR5 in the ABA-deficient mutant aba1-6 (Pro35S:RCAR5/aba1-6; Appendix Fig S2A) and 19

analyzed the relationship between RCAR5-mediated ABA signaling and delayed seed 20

germination under cold stress conditions. At 4°C, the germination rate of aba1-6 seeds was 21

1.3–1.9-fold higher than that of WT seeds; moreover, germinated aba1-6 seedlings had 22

longer radicles than WT seedlings (Fig 3D and E). RCAR5 overexpression did not alter the 23

germination rate of aba1-6 mutants at 4°C. However, similar to Pro35S:RCAR5 seedlings, 24

Pro35S:RCAR5/aba1-6 seedlings were more sensitive to ABA—as measured by lower seed 25


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germination rates and shorter radicles—than were aba1-6 seedlings (Appendix Fig S2B–E). 1

Owing to this ABA hypersensitivity, application of exogenous ABA strongly inhibited 2

germination of Pro35S:RCAR5/aba1-6 seeds under cold stress conditions (Fig 3F). Our data 3

suggest that RCAR5 contributes to cold stress-induced delay of seed germination via an 4

ABA-dependent pathway. 5

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HAB1, but not ABI5, is involved in in delayed germination of Pro35S:RCAR5 seeds 7

under cold stress conditions 8

To examine the mechanism whereby germination of Pro35S:RCAR5 seeds is delayed under 9

cold stress conditions, we genetically analyzed the downstream components of RCAR5 in 10

ABA signaling. Yeast two-hybrid and bimolecular fluorescence complementation assay (BiFC) 11

analyses revealed the interaction of RCAR5 with HAB1 and PP2CA (Appendix Fig S3A and 12

B); RCAR5–HAB1 interaction occurred in the nucleus and cytoplasm, whereas RCAR5–13

PP2CA interaction occurred only in the nucleus. The expression levels of HAB1 and PP2CA 14

in seeds decreased after stratification or imbibition during germination (Appendix Fig S4A 15

and B) and increased in cold stress-treated seeding (Appendix Fig S4C-D). Loss-of-function 16

mutants of HAB1 and PP2CA showed ABA hypersensitivity during germination and seedling 17

growth (Appendix Fig S3C and D) (Lim et al., 2014, Saez et al., 2004a). Consistently, under 18

cold stress conditions, seed germination and seedling growth were more strongly inhibited in 19

hab1 and pp2ca mutants than in WT plants; however, at 16 DAI, the germination rates of 20

hab1 and pp2ca mutants were similar to those of WT plants (Appendix Fig S3C and D). We 21

previously reported that HAB1 acts downstream of RCAR5 in ABA signaling (Lim & Lee, 22

2015); hence, we examined the functional relationship between HAB1 and RCAR5 during 23

seed germination at 4°C. In contrast to hab1 seeds, Pro35S:HAB1 seeds germinated more 24

rapidly than WT seeds (Fig 4A and B). At 13 DAI, the germination rate of 25


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Pro35S:HAB1/Pro35S:RCAR5 seeds did not differ significantly from that of Pro35S:RCAR5 1

seeds; thereafter, the germination rate of Pro35S:HAB1/Pro35S:RCAR5 seeds was 2

significantly higher than that of Pro35S:RCAR5 seeds. Our results indicate that HAB1 3

overexpression compromises RCAR5-induced ABA hypersensitivity in 4

Pro35S:HAB1/Pro35S:RCAR5 seeds; these findings are in agreement with our ABA 5

hyposensitivity data (Appendix Fig S4E-I). 6

Next, we used the ABA-insensitive—abi5 [designated as abi5-8 by Zheng et al. 7

(2012b)]—mutant. As a bZIP transcription factor, ABI5 is mainly expressed in dry seeds and 8

plays a positive role in ABA signaling during seed germination and early seedling growth 9

(Finkelstein & Lynch, 2000, Lopez-Molina et al., 2001). abi5 mutants showed ABA 10

hyposensitivity (Zheng et al., 2012b), whereas ABI5-overexpressing plants were 11

hypersensitive to ABA (Lopez-Molina et al., 2001). We overexpressed RCAR5 in abi5 12

(Pro35S:RCAR5/abi5) mutants; ABA sensitivity—as measured by germination rate and 13

seedling establishment—of these mutants was similar to that of abi5 mutants (Appendix Fig 14

S2F-J). Based on the observed ABA hyposensitivity, we predicted increased germination of 15

abi5 and Pro35S:RCAR5/abi5 seeds under cold stress conditions. However, the germination 16

rate of abi5 seeds was lower than that of WT seeds, although the difference was not 17

significant; moreover, at 18 DAI, abi5 seedlings were smaller than WT seedlings (Fig 4C and 18

D). Pro35S:RCAR5/abi5 seed germination at 4°C was markedly delayed; however, 19

germination rates were significantly higher than those of Pro35S:RCAR5 lines and 20

significantly lower than those of WT seeds (Fig 4D). Our data suggest that cold stress-21

induced delay in the germination of the Pro35S:RCAR5 lines is regulated by HAB1, but not 22

by ABI5. 23

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RCAR5 is induced in leaves in response to cold stress in an ABA-independent 25


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manner 1

To investigate the functional role of RCARs in leaves in response to cold stress, we analyzed 2

expression patterns of RCARs in Arabidopsis leaves in response to cold stress (4°C). In the 3

eFP browser (Winter et al., 2007), several RCARs were downregulated by cold stress; 4

however, no data on RCAR4, RCAR5, RCAR6, or RCAR7 that have much lower expression 5

levels in the leaves relative to the other RCARs were available (Appendix Fig S5A-C). qRT-6

PCR analysis of 14 RCARs revealed cold stress-induced alteration of RCAR gene 7

expression. Consistent with our in-silico data, expression levels of RCAR7, RCAR8, 8

RCAR10, RCAR11, RCAR12, and RCAR13 gradually decreased in response to cold stress, 9

whereas the expression levels of RCAR1, RCAR2, RCAR5, and RCAR6 were strongly 10

induced (>2-fold) relative to the control (Fig 5A). RCAR5 showed the highest induction level 11

(up to 4-fold). ABA deficiency did not alter the expression level of RCAR5 in WT leaves 12

(Appendix Fig S5D). To examine whether cold stress-induced RCAR5 expression occurs in 13

an ABA-independent manner, we analyzed the expression pattern of RCAR5 in the ABA-14

deficient mutants aba1-6 and aba1-3 (Ler background) and the ABA-insensitive mutant abi5 15

under cold stress conditions. Similar to data obtained using the Col-0 ecotype (Fig 5A), the 16

investigated mutants showed gradual expression of RCAR5 in rosette leaves in response to 17

cold stress (Fig 5B), indicating that RCAR5 is induced by cold stress via an ABA-18

independent pathway. 19

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Pro35S:RCAR5 transgenic plants exhibit enhanced stomatal closure in response to 21

cold stress 22

Cold stress triggers leaf wilting and stomatal closure (Ruelland & Zachowski, 2010, 23

Wilkinson et al., 2001). Consistent with our qRT-PCR data (Fig 5B), RCAR5 expression was 24

not induced in leaves treated with ABA, but was strongly induced by dehydration (Appendix 25


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Fig S5E). Hence, RCAR5 expression may be triggered by cold stress-induced leaf 1

dehydration in an ABA-independent manner. In comparison with WT plants, Pro35S:RCAR5 2

plants showed enhanced tolerance to dehydration stress via decreased transpirational water 3

loss from the leaves (Appendix Fig S5F and G) and increased ABA-induced stomatal closure 4

(Lim & Lee, 2015). We conducted phenotypic analysis of Pro35S:RCAR5 plants in response 5

to cold stress. After 24 h incubation at 4°C, WT plants were wilted, but there were no 6

phenotypic differences between Pro35S:RCAR5 plants and non-treated plants (Fig 6A). 7

Interestingly, this phenomenon was observed in Pro35S:RCAR5 plants, but not in 8

Pro35S:RCAR2 or Pro35S:RCAR3 plants (Appendix Fig S6A-C). To monitor this cold stress-9

induced leaf dehydration in a quantitative manner, we measured the fresh weights of aerial 10

parts of WT and Pro35S:RCAR5 plants. In comparison with WT plants, Pro35S:RCAR5 11

plants showed significantly lower water loss; after 24 h incubation at 4°C, the fresh weights 12

of WT and Pro35S:RCAR5 plants were decreased by approximately 44% and 29%, 13

respectively (Fig 6B). To examine whether the decreased transpirational water loss exhibited 14

by Pro35S:RCAR5 plants is derived from rapid stomatal closure in response to cold stress, 15

we measured leaf surface temperature and stomatal apertures. Under normal growth 16

conditions, we observed no phenotypic differences between WT and Pro35S:RCAR5 plants 17

(Fig 6C-F). However, after incubation at 4°C, the leaf temperatures of Pro35S:RCAR5 plants 18

were higher than those of WT plants; after 4 h incubation, the difference in leaf temperature 19

was statistically significant (Fig 6C and D, and Appendix Fig S6D and E). Consistently, the 20

stomatal apertures of Pro35S:RCAR5 plants were significantly smaller than those of WT 21

plants (Fig 6E and F); in comparison with non-treated plants, the average stomatal apertures 22

of WT and Pro35S:RCAR5 plants were reduced by 31% and 40%, respectively. Our data 23

suggest that RCAR5 is involved in cold stress-induced stomatal closure. 24

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Enhanced tolerance of Pro35S:RCAR5 transgenic plants to cold stress is 1

accompanied by high expression levels of cold-responsive genes 2

RCAR5 overexpression conferred enhanced stomatal closure in response to cold stress and 3

ABA; hence, we postulated that Pro35S:RCAR5 plants show enhanced tolerance to freezing 4

stress. We exposed 3-week-old WT and Pro35S:RCAR5 seedlings to freezing temperature 5

(approx. −4 to −6°C) for 1 h. After transfer to 24°C for 2 days, the survival rate of 6

Pro35S:RCAR5 seedlings was higher than that of WT; in comparison with Pro35S:RCAR5 7

seedlings, WT seedlings were severely wilted and did not survive (Fig 6G and H). Under 8

cold stress conditions, the cellular membrane is frequently damaged because of cold stress-9

induced dehydration (Steponkus, 1984, Uemura et al., 1995). The degree of membrane 10

injury in plants can be evaluated by relative electrolyte leakage (Steponkus, 1984). 11

Consistently, our Pro35S:RCAR5 seedlings showed significantly lower electrolyte leakage 12

than WT plants (Fig 6I). 13

Next, we selected 12 representative genes—CBF1–3 (AT4G25490, AT4G25470, 14

AT4G25480), RD29A (AT5G52310), RD29B (AT5G52300), RAB18 (AT5G66400), DREB2A 15

(AT5G05410), COR15A (AT2G42540), COR47 (AT1G20440), RD26 (AT4G27410), KIN1–2 16

(AT3G21960, AT5G15970)—associated with cold stress, dehydration, and ABA signaling. 17

We analyzed the expression patterns of these genes in response to cold stress (4°C). With 18

the exception of DREB2A and COR47, the expression levels of the investigated genes were 19

higher expressed in Pro35S:RCAR5 transgenic lines than in WT plants under normal growth 20

conditions. Moreover, the different expression levels between the two plant lines were still 21

observed after cold stress treatment (Appendix Fig S7A). Our results suggest that high 22

levels of CBF genes during the early stages of cold treatment may help trigger transcription 23

of target genes in Pro35S:RCAR5 transgenic lines. The enhanced gene expression 24

observed in Pro35S:RCAR5 transgenic lines may be derived from ABA hypersensitivity. 25


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RCAR5 overexpression in ABA-insensitive abi5 and Pro35S:HAB1 mutant plants did not 1

lead to a similar expression pattern in response to cold stress (Appendix Fig S7B and C). 2

Our data indicate that upregulation of cold, dehydration, and/or ABA-induced genes may 3

contribute to enhanced tolerance of Pro35S:RCAR5 transgenic lines to cold stress. 4

5

RCAR5 overexpression in aba1-6 mutant seedlings leads to enhanced stomatal 6

closure in response to cold stress 7

ABA is a well-known phytohormone that triggers stomatal closure under abiotic stress 8

conditions such as dehydration (Cutler et al., 2010). To examine whether cold stress-induced 9

enhanced stomatal closure in Pro35S:RCAR5 plants is associated with ABA, we subjected 10

3-week-old WT, Pro35S:RCAR5, aba1-6, and Pro35S:RCAR5/aba1-6 seedlings to cold 11

stress. In comparison with WT plants, aba1-6 and Pro35S:RCAR5/aba1-6 mutant plants 12

wilted rapidly. Thus, it was difficult to distinguish phenotypic differences between 13

Pro35S:RCAR5/aba1-6 and aba1-6 mutant plants in response to cold stress conditions; 14

hence, in further studies, we used only Pro35S:RCAR5/aba1-6 and aba1-6 plant lines. 15

Under normal growth conditions, leaf temperatures were slightly higher in 16

Pro35S:RCAR5/aba1-6 than in aba1-6 plants, but the difference was not significant; however, 17

after cold stress treatment (4°C for 3 h), leaf temperatures were significantly higher in 18

Pro35S:RCAR5/aba1-6 plants than in aba1-6 plants (Fig 7A and B). After a further 3 h of 19

incubation, Pro35S:RCAR5/aba1-6 plants were less wilted than aba1-6 plants (Fig 7C). 20

Similar to Pro35S:RCAR5 plants, this physiological characteristic of Pro35S:RCAR5/aba1-6 21

plants contributed to enhanced survival rates after freezing treatment (−4°C for 1 h) (Fig 7D). 22

However, qRT-PCR analysis revealed that expression levels of the ABA-, dehydration-, and 23

cold-responsive genes RD29B, RAB18, KIN1, COR47, and COR15A did not differ 24

significantly between Pro35S:RCAR5/aba1-6 and aba1-6 plants in response to cold 25


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treatment (4°C for 6 h) (Fig 7E), and this may be derived from ABA deficiency. Our data 1

suggest that cold stress induces stomatal closure via an ABA-independent pathway involving 2

RCAR5. 3

4

Discussion 5

As ABA receptors, Arabidopsis RCARs function redundantly in regulation of seed 6

germination, stomatal aperture, and transcriptional activation in response to ABA (Gonzalez-7

Guzman et al., 2012, Park et al., 2009). Nevertheless, many studies have suggested a 8

distinct function of RCARs in ABA and abiotic stress signaling, based on their different 9

expression patterns, biochemical properties, and genetic analyses data (Antoni et al., 2013, 10

Dupeux et al., 2011, Gonzalez-Guzman et al., 2012, Hao et al., 2011, Zhao et al., 2016, 11

Zhao et al., 2013, Zhao et al., 2014). Our data provide a new insight into the involvement of 12

RCAR5 in cold stress responses, including delay of seed germination and rapid stomatal 13

closure via ABA-dependent and ABA-independent pathways, respectively (Fig 7F). 14

Owing to functional redundancy of the RCAR gene family, we generated transgenic 15

plants overexpressing each of the 14 RCARs that mediate ABA signaling. Overexpression of 16

these RCARs conferred ABA hypersensitivity in terms of seed germination and seedling 17

growth; however, the degree of sensitivity varied according to each Pro35S:RCAR mutant 18

(Appendix Fig S1D–G). Seed dormancy and germination are influenced by ABA content and 19

ABA sensitivity. Several studies have shown that mutations in ABA biosynthesis and 20

signaling components influence the level of seed dormancy. Seed germination occurs more 21

rapidly in ABA-deficient mutants—such as aba1 and aba2—than in WT plants (Gonzalez-22

Guzman et al., 2002, Xiong et al., 2001). Dominant-negative mutants of ABI1 (abi1-1) and 23

ABI2 (abi2) and a mutant defective in three SnRKs (snrk2.2, snrk2.3, and snrk2.6) showed 24

reduced seed dormancy, consistent with their negative and positive roles in ABA signaling, 25


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respectively (Fujita et al., 2009, Ma et al., 2009, Nakashima et al., 2009, Park et al., 2009). 1

Hence, we predicted delayed seed germination of Pro35S:RCAR mutants under cold stress 2

conditions. Consistently, germination of Pro35S:RCAR5 seeds was markedly delayed under 3

cold stress conditions (Fig 2A and B). Germination was also delayed in Pro35S:RCAR2 4

seeds, but less markedly than in Pro35S:RCAR5 seeds. Our data suggest that the depth of 5

seed dormancy under cold stress conditions may be only partly derived from ABA sensitivity. 6

This discrepancy between ABA sensitivity and seed dormancy was also observed in the 7

loss-of-function mutant of ABI5 (abi5; Fig 4C and D, and Appendix Fig S2F-J). ABI5 does not 8

control seed dormancy, but does regulate seed germination and seedling growth in response 9

to ABA (Finkelstein & Lynch, 2000, Lopez-Molina et al., 2001). Consistently, the germination 10

rate of abi5 seeds at 4°C did not differ significantly from that of WT seeds; moreover, similar 11

to Pro35S:RCAR5 seeds, germination of Pro35S:RCAR5/abi5 seeds at 4°C was markedly 12

delayed by RCAR5 overexpression. However, these data do not imply that Pro35S:RCAR5 13

seeds can be slowly germinated through an ABA-independent pathway. Germination of 14

Pro35S:RCAR5 seeds under cold stress conditions was dependent on endogenous ABA. 15

RCAR5-mediated seed dormancy was blocked by ABA deficiency derived from NF treatment 16

and loss-of-function of ABA1 (Fig 3) and was regulated by HAB1—a downstream component 17

of RCAR5 in ABA signaling (Fig 4A and B). In addition, dry and imbibed Pro35S:RCAR5 18

seeds showed high expression levels of ABA-responsive genes, suggesting that germination 19

was delayed by enhanced ABA signaling (Fig 2D). Consistently, RCAR5 expression in dry 20

seeds was ABA dependent and decreased significantly during seed germination (Fig 1A and 21

C). Consistent with RCAR5, transcripts of RCAR1, RCAR2, and RCAR6 accumulated 22

strongly in dry seeds—regardless of transcriptional difference between Col-0 and Ler 23

ecotypes—and were downregulated during seed germination. However, the effect of gene 24

overexpression on seed germination under cold stress conditions occurred infrequently. Our 25


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data suggest that RCAR5 plays a major role in the maintenance of ABA-induced seed 1

dormancy under cold stress conditions. 2

In contrast to the expression pattern in dry seeds, RCAR5 expression in leaf tissues was 3

much weaker than that of other RCAR gene family members and was not influenced by ABA 4

deficiency (Fig 5C and D). Nevertheless, RCAR5 expression was significantly upregulated in 5

response to cold stress, whereas expression of RCAR7, RCAR8, RCAR10, RCAR11, 6

RCAR12, and RCAR13 was downregulated (Fig 5A). RCAR5 expression was also induced 7

via an ABA-independent pathway: Under cold-stress conditions, ABA-deficient aba1-6 and 8

ABA-insensitive abi5 mutants showed similar RCAR5 expression patterns to WT plants (Fig 9

5B). Moreover, RCAR5 expression was influenced by dehydration, but not by ABA treatment 10

(Appendix Fig S5E). Some RCARs—including RCAR1, RCAR3, and RCAR10—inhibit 11

PP2Cs in the absence of ABA (Hao et al., 2011). Hence, we predicted that RCAR5 functions 12

in plant cold stress responses via an ABA-independent pathway. Pro35S:RCAR5 plants 13

showed enhanced stomatal closure in response to cold stress (Fig 6). Our results are 14

consistent with those of Wilkinson et al. (2001), who reported rapidly induced stomatal 15

closure in the leaves of Commelina communis in response to cold stress, possibly owing to 16

increased uptake of apoplastic calcium, but not ABA, into guard cells. This phenomenon 17

seems to be specific to Pro35S:RCAR5 plants; ABA-hypersensitive Pro35S:RCAR2 and 18

Pro35S:RCAR3 mutants did not show enhanced stomatal closure in response to cold stress 19

(Appendix Fig S6A–C). We further showed that leaf temperatures of loss-of-function mutants 20

of HAB1 and PP2CA—which can interact with RCAR5—did not differ significantly from those 21

of WT plants in response to cold stress (Appendix Fig S3E-H). However, we cannot rule out 22

the involvement of a pleiotropic effect of Pro35S:RCAR5 plants and functional redundancy of 23

HAB1 and PP2CA. We questioned whether cold-stress induced rapid stomatal closure in 24

Pro35S:RCAR5 plants occurs via an ABA-independent pathway. Pro35S:RCAR5 plants 25


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19

showed ABA hypersensitivity and ABA acts as the main signal for triggering stomatal closure, 1

hence, the involvement of ABA in the process remains unclear. Previous studies have 2

suggested that cold stress triggers ABA biosynthesis and that ABA plays an important role in 3

the cold stress response—in particular cold acclimation, as shown in ABA-deficient and ABA-4

insensitive mutants (Kushiro et al., 2004, Lång et al., 1994, Thomashow, 1999, Xiong et al., 5

2001). For example, ABA-deficient mutants aba1 and aba3 showed impaired freezing 6

tolerance characterized by low expression levels of ABA-responsive genes that may function 7

in cold stress (Xiong et al., 2001). Additionally, application of exogenous ABA triggers cold 8

acclimation and enhanced freezing tolerance in Arabidopsis (Thomashow, 1999). However, 9

ABA biosynthesis seems not to be an early event in the cold stress response. In Arabidopsis, 10

endogenous ABA accumulated weakly at 6 h, and its level increased up to 4-fold after 15 h 11

exposure to 4°C day/2°C night temperatures (Lång et al., 1994). Moreover, expression of 12

ABA synthesis-related genes was not upregulated at 3 h, 6 h, or 24 h after exposure to 0°C 13

(Lee et al., 2005). Here, surface temperature and fresh weight of leaves differed significantly 14

between Pro35S:RCAR5 and WT plants 4 h after cold treatment. In comparison with the 15

timing of ABA biosynthesis, stomatal closure of Pro35S:RCAR5 plants seems to start earlier. 16

In addition, RCAR5 overexpression in aba1-6 mutants led to enhanced stomatal closure in 17

response to cold stress. Our data imply that RCAR5 is induced in response to cold stress 18

and contributes to cold stress-induced stomatal closure via an ABA-independent pathway. 19

Thus, our study provides evidence for two different functions of RCAR5 via ABA-20

dependent and ABA-independent pathways in plant tissues such as seeds and leaves. 21

However, the precise function of RCAR5 in the cold stress response remains unclear. 22

Further studies to elucidate the functional interactions RCAR5–HAB1 and PP2CA–OST1 in 23

germination delay and rapid stomatal closure under cold stress conditions, and to identify 24

transcription factors that regulate RCAR5 expression via ABA-dependent and/or ABA-25


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20

independent pathways, are required. 1

2

Materials and Methods 3

Plant material and growth conditions 4

Here, Arabidopsis thaliana (ecotype Col-0 and Ler) plants were used as the wild-type (WT). 5

The T-DNA insertion mutants abi5 (SALK_013163) (Zheng et al., 2012a), hab1 6

(SALK_002104) (Saez et al., 2004b), and pp2ca-1 (SALK_124564; Lim et al., 2014), and the 7

EMS-mutagenized mutant aba1-3 (Ler background) (Koornneef et al., 1982) (Koornneef, 8

Jorna et al., 1982) 62 61 and aba1-6 (Col-0 background) , were obtained from the 9

Arabidopsis Biological Resource Center. Plants were routinely grown in soil mixture 10

containing a 9:1:1 ratio of peat moss, perlite and vermiculite. The plants were maintained at 11

24°C and 60% humidity under fluorescent light (130 μmol photons·m-2·s-1) with a 16 h light/8 12

h dark cycle. Prior to in vitro culture, seeds of Arabidopsis were surface sterilized with 70% 13

ethanol for 1 min and treated with 2% sodium hydroxide for 10 min. The seeds were then 14

washed 10 times with sterile distilled water and sown on MS agar plates (Sigma, St. Louis, 15

MO) supplemented with 1% sucrose. Following stratification at 4°C for 2 days, the plates 16

were sealed and incubated at 24°C in a chamber under fluorescent light (130 μmol 17

photons·m-2·s-1) with a 16 h light/8 h dark cycle. For tobacco (Nicotiana benthamiana) plants, 18

seeds were sown in a steam-sterilized compost soil mix (peat moss, perlite, and vermiculite, 19

5:3:2, v/v/v), sand, and loam soil (1:1:1, v/v/v). The tobacco plants were raised in a growth 20

chamber at 25 ± 1°C under the conditions described above. 21

22

Generation of transgenic RCAR -overexpressing mutants 23

We previously generated Arabidopsis transgenic lines overexpressing RCAR2 (Lee et al., 24

2013), RCAR3 (Lim et al., 2014), RCAR4, and RCAR5 (Lim & Lee, 2015). Here, we 25


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21

generated transgenic lines overexpressing each of the remaining RCAR gene family 1

members in Arabidopsis. The coding sequences of the 10 RCARs were cloned into the 2

pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA, USA) and integrated into pK2GW7 using 3

the LR reaction to induce constitutive expression of each RCAR under the control of the 4

cauliflower mosaic virus 35S promoter (Karimi et al., 2002). The correct construct was 5

introduced into Agrobacterium tumefaciens strain GV3101 via electroporation. We 6

conducted Agrobacterium-mediated transformation using the floral dip method (Clough & 7

Bent, 1998). For selection of transgenic lines, seeds harvested from the putative transformed 8

plants were plated on MS agar plates containing 50 μg ml–1 kanamycin. 9

10

Cold treatment and phenotypic analyses 11

To measure the germination rate of WT and transgenic plants under cold stress conditions, 12

seeds were vernalized at 4°C for 2 days and continuously incubated in darkness until the 13

indicated time points. To analyze whether cold sensitivity of Arabidopsis plants is associated 14

with ABA signaling, seeds were plated on MS agar medium supplemented with ABA (0.75 15

μM) and/or norflurazon (NF; 25 μM or 50 μM) as the ABA biosynthesis inhibitor. 16

For freezing tolerance assays, 3-week-old Arabidopsis seedlings were exposed to 4 ± 17

1°C for 0.5 h, followed by 1°C for 1 h. To set experimental temperatures, the temperature 18

was incrementally decreased by 2°C over a period of 30 min and maintained for 1 h. After 19

freezing treatment, plants were incubated at 4 ± 1°C in the dark for 12 h and then transferred 20

to normal growth conditions. The survival rate after 4 days was measured. 21

For thermal imaging analysis, 2-week-old Arabidopsis seedlings were subjected to cold 22

stress by exposure to 4 ± 1°C until the indicated time points. Thermal images were obtained 23

using an infrared camera (FLIR systems; T420), and leaf temperatures were measured with 24

FLIR Tools+ version 5.2 software. 25


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22

1

Electrolyte leakage test 2

Electrolyte leakage of WT and Pro35S:RCAR5 transgenic plants subjected to freezing stress 3

was measured as described previously, but with modifications to sample type and incubation 4

time (Ding et al., 2015). For cold acclimation, 2-week-old plants grown in soil were treated at 5

4 ± 1°C for 2 days and then exposed to −4°C for 1 h prior to an electrolyte leakage test. Leaf 6

samples were detached from each plant line and placed into tubes containing 20 mL of 7

deionized water (S0). Following shaking for 12 h at room temperature, the conductivity of the 8

samples was measured (S1). After autoclaving for 15 min, the tubes were shaken for 1 h and 9

the conductivity was measured (S2). Electrolyte leakage of each sample was calculated 10

using the formula S1 – S0/S2 – S0. 11

12

RNA isolation and quantitative reverse transcription-polymerase chain reaction 13

Total RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) analyses 14

were performed as described previously (Lim & Lee, 2016). Two-week-old WT and 15

transgenic plants were subjected to cold stress (4°C) and leaf samples were harvested at 16

the indicated time points. cDNA was synthesized using a Transcript First Strand cDNA 17

Synthesis kit (Roche, Indianapolis, IN) with 1 µg of total RNA according to the 18

manufacturer’s instructions. For quantitative RT-PCR (qRT-PCR) analysis, the synthesized 19

cDNA was amplified in a CFX96 TouchTM Real-Time PCR detection system (Bio-Rad, 20

Hercules, CA) with iQTMSYBR Green Supermix and specific primers (Appendix Table 1). All 21

reactions were performed in triplicate. The relative expression level of each gene was 22

calculated using the ∆∆Ct method, as described previously (Livak & Schmittgen, 2001). The 23

Arabidopsis actin8 gene (AtACT8) was used for normalization. 24

25


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23

Stomatal aperture bioassay 1

The stomatal aperture bioassay was conducted as described previously, but with some 2

modifications (Lim et al., 2014). Briefly, leaf peels were collected from the rosette leaves of 3

3-week-old plants and floated on stomatal opening solution (SOS; 50 mM KCl, 10 mM MES-4

KOH, 10 μM CaCl2, pH 6.15). The peels were incubated at 24°C under fluorescent light for 3 5

h to obtain >80% stomatal opening in Arabidopsis thaliana Col-0 plants. The buffer was 6

replaced with fresh SOS and leaf peels were further incubated at 4°C under fluorescent light 7

for 3 h. In each individual sample, 100 stomata were randomly observed under a Nikon 8

Eclipse 80i microscope. The widths and lengths of individual stomatal pores were recorded 9

using Image J 1.46r software (http://imagej.nih.gov/ij). Each experiment was performed in 10

triplicate. 11

12

Yeast two-hybrid assay 13

Yeast two-hybrid analysis was performed as described previously (Lim & Lee, 2016). The 14

coding sequences of RCAR5 as bait or ABI1, HAB1, AHG1, and PP2CA as prey were 15

subcloned into pGBKT7 or pGADT7, respectively. Using the lithium acetate-mediated 16

transformation method, these bait and prey pairs were co-transformed into yeast strain 17

AH109. To evaluate the interaction between them, transformant candidates were screened 18

on SC-Leu-Trp and SC-Ade-His-Leu-Trp media. 19

20

Bimolecular fluorescence complementation assay 21

The bimolecular fluorescence complementation (BiFC) assay was performed as described 22

previously (Lim & Lee, 2015). To generate the BiFC constructs, the coding sequences of 23

RCAR5, HAB1, and PP2CA were subcloned into the Pro35S:VYNE and Pro35S:CYCE 24

vectors using the LR reaction (Waadt et al., 2008). The resulting plasmids were transformed 25


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24

into Agrobacterium tumefaciens strain GV3101. For agroinfiltration-based transient gene 1

expression, Agrobacterium tumefaciens harboring RCAR5:VYNE combined with either 2

HAB1:CYCE or PP2CA:CYCE were syringe-infiltrated into the leaves of 5-week-old 3

Nicotiana benthamiana seedlings together with the silencing suppressor p19 in a 1:1:1 4

volume ratio at a final OD600 = 0.5 for each. At 3 days after infiltration, leaf discs were cut and 5

the lower epidermal cells were examined under a confocal microscope (510 UV/Vis Meta; 6

Zeiss, Oberkochen, Germany) equipped with LSM Image Browser software. 7

8

Acknowledgments 9

This work was supported by a grant from the “Next-Generation BioGreen 21 Program for 10

Agriculture & Technology Development (Project No. PJ01316801),” Rural Development 11

Administration, and by the National Research Foundation of Korea (NRF) grant funded by 12

the Korea Government (MSIT) (No. 2018R1A5A1023599, SRC), Republic of Korea. 13

14

Author Contributions 15

C.W.L and S.C.L conceptualized and designed the study. C.W.L performed all the 16

experiments. S.C.L supervised the project. C.W.L and S.C.L wrote the manuscript. 17

18

Declaration of Interests 19

The authors declare no competing interests. 20

21

References 22

Antoni R, Gonzalez-Guzman M, Rodriguez L, et al (2013) PYRABACTIN RESISTANCE1-23

LIKE8 plays an important role for the regulation of abscisic acid signaling in root. Plant 24

Physiol 161: 931-941 25


https://doi.org/10.1101/723627

25

Baskin JM, Baskin CC (2004) A classification system for seed dormancy. Seed Sci Res 14: 1

1-16 2

Bhaskara GB, Nguyen TT, Verslues PE (2012) Unique Drought Resistance Functions of the 3

Highly ABA-Induced Clade A Protein Phosphatase 2Cs. Plant Physiol 160: 379-395 4

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated 5

transformation of Arabidopsis thaliana. The Plant Journal 16: 735-743 6

Cuevas JC, Lopez-Cobollo R, Alcazar R, Zarza X, Koncz C, Altabella T, Salinas J, Tiburcio 7

AF, Ferrando A (2008) Putrescine is involved in Arabidopsis freezing tolerance and cold 8

acclimation by regulating abscisic acid levels in response to low temperature. Plant 9

Physiol 148: 1094-1105 10

Cutler SR, Rodriguez PL, Finkelstein RR, Abrams SR (2010) Abscisic acid: emergence of a 11

core signaling network. Annu Rev Plant Biol 61: 651-79 12

Ding Y, Li H, Zhang X, Xie Q, Gong Z, Yang S (2015) OST1 kinase modulates freezing 13

tolerance by enhancing ICE1 stability in Arabidopsis. Developmental Cell 32: 278-89 14

Dupeux F, Santiago J, Betz K, Twycross J, Park SY, Rodriguez L, Gonzalez-Guzman M, 15

Jensen MR, Krasnogor N, Blackledge M, Holdsworth M, Cutler SR, Rodriguez PL, 16

Marquez JA (2011) A thermodynamic switch modulates abscisic acid receptor sensitivity. 17

Embo J 30: 4171-4184 18

Finkelstein R, Reeves W, Ariizumi T, Steber C (2008) Molecular aspects of seed dormancy. 19

Annual Review of Plant Biology 59: 387-415 20

Finkelstein RR, Lynch TJ (2000) The arabidopsis abscisic acid response gene ABI5 encodes 21

a basic leucine zipper transcription factor. Plant Cell 12: 599-609 22

Fujii H, Chinnusamy V, Rodrigues A, Rubio S, Antoni R, Park SY, Cutler SR, Sheen J, 23

Rodriguez PL, Zhu JK (2009) In vitro reconstitution of an abscisic acid signalling 24

pathway. Nature 462: 660-4 25


https://doi.org/10.1101/723627

26

Fujita Y, Nakashima K, Yoshida T, Katagiri T, Kidokoro S, Kanamori N, Umezawa T, Fujita M, 1

Maruyama K, Ishiyama K, Kobayashi M, Nakasone S, Yamada K, Ito T, Shinozaki K, 2

Yamaguchi-Shinozaki K (2009) Three SnRK2 Protein Kinases are the Main Positive 3

Regulators of Abscisic Acid Signaling in Response to Water Stress in Arabidopsis. Plant 4

and Cell Physiology 50: 2123-2132 5

Furihata T, Maruyama K, Fujita Y, Umezawa T, Yoshida R, Shinozaki K, Yamaguchi-6

Shinozaki K (2006) Abscisic acid-dependent multisite phosphorylation regulates the 7

activity of a transcription activator AREB1. P Natl Acad Sci USA 103: 1988-1993 8

Geiger D, Scherzer S, Mumm P, Stange A, Marten I, Bauer H, Ache P, Matschi S, Liese A, 9

Al-Rasheid KAS, Romeis T, Hedrich R (2009) Activity of guard cell anion channel 10

SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. P Natl Acad 11

Sci USA 106: 21425-21430 12

Gilmour SJ, Thomashow MF (1991) Cold-Acclimation and Cold-Regulated Gene-Expression 13

in Aba Mutants of Arabidopsis-Thaliana. Plant Mol Biol 17: 1233-1240 14

Gonzalez-Guzman M, Apostolova N, Belles JM, Barrero JM, Piqueras P, Ponce MR, Micol 15

JL, Serrano R, Rodriguez PL (2002) The short-chain alcohol dehydrogenase ABA2 16

catalyzes the conversion of xanthoxin to abscisic aldehyde. Plant Cell 14: 1833-1846 17

Gonzalez-Guzman M, Pizzio GA, Antoni R, Vera-Sirera F, Merilo E, Bassel GW, Fernandez 18

MA, Holdsworth MJ, Perez-Amador MA, Kollist H, Rodriguez PL (2012) Arabidopsis 19

PYR/PYL/RCAR receptors play a major role in quantitative regulation of stomatal 20

aperture and transcriptional response to abscisic acid. Plant Cell 24: 2483-96 21

Hao Q, Yin P, Li WQ, Wang L, Yan CY, Lin ZH, Wu JZ, Wang JW, Yan SF, Yan N (2011) The 22

Molecular Basis of ABA-Independent Inhibition of PP2Cs by a Subclass of PYL Proteins. 23

Mol Cell 42: 662-672 24

Hubbard KE, Nishimura N, Hitomi K, Getzoff ED, Schroeder JI (2010) Early abscisic acid 25


https://doi.org/10.1101/723627

27

signal transduction mechanisms: newly discovered components and newly emerging 1

questions. Genes Dev 24: 1695-708 2

Karimi M, Inze D, Depicker A (2002) GATEWAY((TM)) vectors for Agrobacterium-mediated 3

plant transformation. Trends Plant Sci 7: 193-195 4

Kilian J, Whitehead D, Horak J, Wanke D, Weinl S, Batistic O, D'Angelo C, Bornberg-Bauer 5

E, Kudla J, Harter K (2007) The AtGenExpress global stress expression data set: 6

protocols, evaluation and model data analysis of UV-B light, drought and cold stress 7

responses. Plant J 50: 347-363 8

Klepikova AV, Kasianov AS, Gerasimov ES, Logacheva MD, Penin AA (2016) A high 9

resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-10

seq profiling. Plant J 88: 1058-1070 11

Knight H, Zarka DG, Okamoto H, Thomashow ME, Knight MR (2004) Abscisic acid induces 12

CBF gene transcription and subsequent induction of cold-regulated genes via the CRT 13

promoter element. Plant Physiol 135: 1710-1717 14

Koornneef M, Jorna ML, Derswan DLCB, Karssen CM (1982) The Isolation of Abscisic-Acid 15

(Aba) Deficient Mutants by Selection of Induced Revertants in Non-Germinating 16

Gibberellin Sensitive Lines of Arabidopsis-Thaliana (L) Heynh. Theor Appl Genet 61: 17

385-393 18

Kreps JA, Wu YJ, Chang HS, Zhu T, Wang X, Harper JF (2002) Transcriptome changes for 19

Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol 130: 2129-2141 20

Kushiro T, Okamoto M, Nakabayashi K, Yamagishi K, Kitamura S, Asami T, Hirai N, Koshiba 21

T, Kamiya Y, Nambara E (2004) The Arabidopsis cytochrome P450CYP707A encodes 22

ABA 8 '-hydroxylases: key enzymes in ABA catabolism. Embo J 23: 1647-1656 23

Lång V, Mäntylä E, Welin B, Sundberg B, Palva ET (1994) Alterations in Water Status, 24

Endogenous Abscisic-Acid Content, and Expression of Rab18 Gene during the 25


https://doi.org/10.1101/723627

28

Development of Freezing Tolerance in Arabidopsis-Thaliana. Plant Physiol 104: 1341-1

1349 2

Lee BH, Henderson DA, Zhu JK (2005) The Arabidopsis cold-responsive transcriptome and 3

its regulation by ICE1. Plant Cell 17: 3155-3175 4

Lee HG, Seo PJ (2015) The MYB96-HHP module integrates cold and abscisic acid signaling 5

to activate the CBF-COR pathway in Arabidopsis. Plant J 82: 962-977 6

Lee SC, Lan WZ, Buchanan BB, Luan S (2009) A protein kinase-phosphatase pair interacts 7

with an ion channel to regulate ABA signaling in plant guard cells. P Natl Acad Sci USA 8

106: 21419-21424 9

Lee SC, Lim CW, Lan W, He K, Luan S (2013) ABA signaling in guard cells entails a dynamic 10

protein-protein interaction relay from the PYL-RCAR family receptors to ion channels. 11

Mol Plant 6: 528-38 12

Lee SJ, Kang JY, Park HJ, Kim MD, Bae MS, Choi HI, Kim SY (2010) DREB2C Interacts 13

with ABF2, a bZIP Protein Regulating Abscisic Acid-Responsive Gene Expression, and 14

Its Overexpression Affects Abscisic Acid Sensitivity. Plant Physiol 153: 716-727 15

Lim CW, Lee SC (2015) Arabidopsis abscisic acid receptors play an important role in 16

disease resistance. Plant Mol Biol 88: 313-324 17

Lim CW, Lee SC (2016) Pepper protein phosphatase type 2C, CaADIP1 and its interacting 18

partner CaRLP1 antagonistically regulate ABA signalling and drought response. Plant 19

Cell and Environment 39: 1559-1575 20

Lim CW, Luan S, Lee SC (2014) A prominent role for RCAR3-mediated ABA signaling in 21

response to Pseudomonas syringae pv. tomato DC3000 infection in Arabidopsis. Plant 22

and Cell Physiology 55: 1691-703 23

Liu X, Hu PW, Huang MK, Tang Y, Li YG, Li L, Hou XL (2016) The NF-YC-RGL2 module 24

integrates GA and ABA signalling to regulate seed germination in Arabidopsis. Nat 25


https://doi.org/10.1101/723627

29

Commun 7 1

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time 2

quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-8 3

Lopez-Molina L, Mongrand S, Chua NH (2001) A postgermination developmental arrest 4

checkpoint is mediated by abscisic acid and requires the AB15 transcription factor in 5

Arabidopsis. P Natl Acad Sci USA 98: 4782-4787 6

Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E (2009) Regulators of 7

PP2C phosphatase activity function as abscisic acid sensors. Science 324: 1064-8 8

Millar AA, Jacobsen JV, Ross JJ, Helliwell CA, Poole AT, Scofield G, Reid JB, Gubler F 9

(2006) Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 10

8'-hydroxylase. Plant J 45: 942-954 11

Müller K, Tintelnot S, Leubner-Metzger G (2006) Endosperm-limited Brassicaceae seed 12

germination: Abscisic acid inhibits embryo-induced endosperm weakening of Lepidium 13

sativum (cress) and endosperm rupture of cress and Arabidopsis thaliana. Plant and 14

Cell Physiology 47: 864-877 15

Nakashima K, Fujita Y, Kanamori N, Katagiri T, Umezawa T, Kidokoro S, Maruyama K, 16

Yoshida T, Ishiyama K, Kobayashi M, Shinozaki K, Yamaguchi-Shinozaki K (2009) 17

Three Arabidopsis SnRK2 Protein Kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 18

and SRK2I/SnRK2.3, Involved in ABA Signaling are Essential for the Control of Seed 19

Development and Dormancy. Plant and Cell Physiology 50: 1345-1363 20

Nishimura N, Sarkeshik A, Nito K, Park SY, Wang A, Carvalho PC, Lee S, Caddell DF, Cutler 21

SR, Chory J, Yates JR, Schroeder JI (2010) PYR/PYL/RCAR family members are major 22

in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis. Plant J 61: 290-23

299 24

Niyogi KK, Grossman AR, Bjorkman O (1998) Arabidopsis mutants define a central role for 25


https://doi.org/10.1101/723627

30

the xanthophyll cycle in the regulation of photosynthetic energy conversion. Plant Cell 1

10: 1121-34 2

Park SY, Fung P, Nishimura N, et al (2009) Abscisic acid inhibits type 2C protein 3

phosphatases via the PYR/PYL family of START proteins. Science 324: 1068-1071 4

Ruelland E, Zachowski A (2010) How plants sense temperature. Environ Exp Bot 69: 225-5

232 6

Saez A, Apostolova N, Gonzalez-Guzman M, Gonzalez-Garcia MP, Nicolas C, Lorenzo O, 7

Rodriguez PL (2004a) Gain-of-function and loss-of-function phenotypes of the protein 8

phosphatase 2C HAB1 reveal its role as a negative regulator of abscisic acid signalling. 9

Plant J 37: 354-369 10

Saez A, Apostolova N, Gonzalez-Guzman M, Gonzalez-Garcia MP, Nicolas C, Lorenzo O, 11

Rodriguez PL (2004b) Gain-of-function and loss-of-function phenotypes of the protein 12

phosphatase 2C HAB1 reveal its role as a negative regulator of abscisic acid signalling. 13

Plant J 37: 354-69 14

Santiago J, Rodrigues A, Saez A, Rubio S, Antoni R, Dupeux F, Park SY, Marquez JA, Cutler 15

SR, Rodriguez PL (2009) Modulation of drought resistance by the abscisic acid receptor 16

PYL5 through inhibition of clade A PP2Cs. Plant J 60: 575-588 17

Seo M, Hanada A, Kuwahara A, Endo A, Okamoto M, Yamauchi Y, North H, Marion-Poll A, 18

Sun TP, Koshiba T, Kamiya Y, Yamaguchi S, Nambara E (2006) Regulation of hormone 19

metabolism in Arabidopsis seeds: phytochrome regulation of abscisic acid metabolism 20

and abscisic acid regulation of gibberellin metabolism. Plant J 48: 354-366 21

Shu K, Liu XD, Xie Q, He ZH (2016) Two Faces of One Seed: Hormonal Regulation of 22

Dormancy and Germination. Molecular Plant 9: 34-45 23

Steponkus PL (1984) Role of the Plasma-Membrane in Freezing-Injury and Cold-Acclimation. 24

Annu Rev Plant Phys 35: 543-584 25


https://doi.org/10.1101/723627

31

Sun S, Fan W, Mu Z (2017) The spatio-temporal specificity of PYR1/PYL/RCAR ABA 1

receptors in response to developmental and environmental cues. Plant signaling & 2

behavior 12: e1214793 3

Szostkiewicz I, Richter K, Kepka M, Demmel S, Ma Y, Korte A, Assaad FF, Christmann A, 4

Grill E (2010) Closely related receptor complexes differ in their ABA selectivity and 5

sensitivity. Plant J 61: 25-35 6

Thomashow MF (1999) Plant cold acclimation: Freezing tolerance genes and regulatory 7

mechanisms. Annu Rev Plant Phys 50: 571-599 8

Tischer SV, Wunschel C, Papacek M, Kleigrewe K, Hofmann T, Christmann A, Grill E (2017) 9

Combinatorial interaction network of abscisic acid receptors and coreceptors from 10

Arabidopsis thaliana. P Natl Acad Sci USA 114: 10280-10285 11

Uemura M, Joseph RA, Steponkus PL (1995) Cold-Acclimation of Arabidopsis-Thaliana - 12

Effect on Plasma-Membrane Lipid-Composition and Freeze-Induced Lesions. Plant 13

Physiol 109: 15-30 14

Waadt R, Schmidt LK, Lohse M, Hashimoto K, Bock R, Kudla J (2008) Multicolor bimolecular 15

fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK 16

complexes in planta. Plant J 56: 505-16 17

Wilkinson S, Clephan AL, Davies WJ (2001) Rapid low temperature-induced stomatal 18

closure occurs in cold-tolerant Commelina communis leaves but not in cold-sensitive 19

tobacco leaves, via a mechanism that involves apoplastic calcium but not abscisic acid. 20

Plant Physiol 126: 1566-1578 21

Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007) An "Electronic 22

Fluorescent Pictograph" Browser for Exploring and Analyzing Large-Scale Biological 23

Data Sets. Plos One 2 24

Xing L, Zhao Y, Gao JH, Xiang CB, Zhu JK (2016) The ABA receptor PYL9 together with 25


https://doi.org/10.1101/723627

32

PYL8 plays an important role in regulating lateral root growth. Sci Rep-Uk 6 1

Xiong LM, Ishitani M, Lee H, Zhu JK (2001) The Arabidopsis LOS5/ABA3 locus encodes a 2

molybdenum cofactor sulfurase and modulates cold stress- and osmotic stress-3

responsive gene expression. Plant Cell 13: 2063-2083 4

Zentella R, Zhang ZL, Park M, Thomas SG, Endo A, Murase K, Fleet CM, Jikumaru Y, 5

Nambara E, Kamiya Y, Sun TP (2007) Global analysis of DELLA direct targets in early 6

gibberellin signaling in Arabidopsis. Plant Cell 19: 3037-3057 7

Zhao Y, Chan ZL, Gao JH, Xing L, Cao MJ, Yu CM, Hu YL, You J, Shi HT, Zhu YF, Gong YH, 8

Mu ZX, Wang HQ, Deng X, Wang PC, Bressan RA, Zhu JK (2016) ABA receptor PYL9 9

promotes drought resistance and leaf senescence. P Natl Acad Sci USA 113: 1949-10

1954 11

Zhao Y, Chan ZL, Xing L, Liu XD, Hou YJ, Chinnusamy V, Wang PC, Duan CG, Zhu JK 12

(2013) The unique mode of action of a divergent member of the ABA-receptor protein 13

family in ABA and stress signaling. Cell Res 23: 1380-1395 14

Zhao Y, Xing L, Wang XG, Hou YJ, Gao JH, Wang PC, Duan CG, Zhu XH, Zhu JK (2014) 15

The ABA Receptor PYL8 Promotes Lateral Root Growth by Enhancing MYB77-16

Dependent Transcription of Auxin-Responsive Genes. Sci Signal 7 17

Zheng Y, Schumaker KS, Guo Y (2012a) Sumoylation of transcription factor MYB30 by the 18

small ubiquitin-like modifier E3 ligase SIZ1 mediates abscisic acid response in 19

Arabidopsis thaliana. Proc Natl Acad Sci U S A 109: 12822-7 20

Zheng Y, Schumaker KS, Guo Y (2012b) Sumoylation of transcription factor MYB30 by the 21

small ubiquitin-like modifier E3 ligase SIZ1 mediates abscisic acid response in 22

Arabidopsis thaliana. P Natl Acad Sci USA 109: 12822-12827 23

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Figure legends 1

Figure 1 RCAR expression in Arabidopsis dry seeds and during seed germination 2

under cold stress conditions. 3

A Differential RCAR expression in dry seeds of ABA-deficient mutants aba1-6 (Col-0 4

background; upper) and aba1-3 (Ler background; bottom). PP2A was used as an internal 5

control for normalization. The expression level of each RCAR in wild-type (WT) Arabidopsis 6

seeds was set to 1.0. ABA-responsive gene ABI5 and MFT were used as positive controls. 7

B Germination rate of WT (Col-0) seeds under cold stress (4°C) conditions. For germination 8

rate, seeds with radicle protrusion were counted as germinated. 9

C RCAR expression during seed germination under cold stress conditions. ABA-responsive 10

gene ABI5 and cell wall-related gene EXP3 were used as controls for seed germination. All 11

data represent mean ± standard deviation (SD) of three independent experiments. Asterisks 12

and different letters indicate significant difference between samples (Student’s t-test for A 13

and ANOVA test for C, P < 0.05). 14

15

Figure 2 Delayed germination of Pro35S:RCAR5 seeds under cold stress conditions. 16

A Seedling development of Pro35S:RCAR transgenic lines and WT plants exposed to cold 17

stress and ABA. Seeds of Pro35S:RCAR transgenic lines were germinated on 0.5 MS 18

medium supplemented with 0 μM or 0.5 μM ABA and vertically grown at 8°C in the dark (left) 19

and at 24°C in the light (middle and right). Representative images were taken at the 20

indicated time points. DAI, days after incubation. 21

B Germination and seedling growth of Pro35S:RCAR mutants under cold stress conditions. 22

For measuring germination rates and seedling growth of each plant line, the numbers of 23


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germinated seeds (with an emerged radicle) and etiolated seedlings were counted at 13 DAI 1

and 21 DAI, respectively. 2

C Recovery of delayed germination of Pro35S:RCAR5 seeds under normal growth 3

conditions. After incubation for 18 days at 4°C in the dark, Pro35S:RCAR5 seeds were 4

transferred to 24°C with light. Representative images were taken at the indicated time points. 5

D Expression patterns of ABA-responsive genes in dry seeds of Pro35S:RCAR mutants. 6

E Expression patterns of ABA-responsive genes and cell-wall related genes in seeds of 7

Pro35S:RCAR mutants and WT plants during germination at 4°C in the dark. All data 8

represent mean ± SD of three independent experiments. Asterisks and different letters 9

indicate significant difference compared with WT (Student’s t-test for B and ANOVA test for D 10

and E; *P < 0.05; **P < 0.01). 11

12

Figure 3 Cold-induced germination delay of Pro35S:RCAR5 seeds in an ABA-13

dependent manner. 14

A-C Effect of norflurazon on seed germination of WT and Pro35S:RCAR5 lines. Seeds of 15

Pro35S:RCAR5 and WT were germinated on 0.5 MS medium supplemented with 16

norflurazon (25 μM or 50 μM) and/or ABA (0.75 μM) and vertically grown at 4°C in the dark. 17

After 24 days, representative images were taken (A). For measuring germination rates of 18

each plant line, the numbers of seeds with emerged radicles were counted at the indicated 19

time points (B and C). 20

D-F Germination and seedling growth of Pro35S:RCAR/aba1-6 mutants under cold stress 21

conditions. Seeds of each transgenic line were plated on 0.5 MS medium supplemented 22

with 0 μM or 0.75 μM ABA and germinated in the dark at 4°C. Representative images were 23


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taken at 16 DAI (D) and germination rate was measured at the indicated time points (E and 1

F). All data represent mean ± SD of three independent experiments. Different letters indicate 2

significant difference compared with WT (ANOVA; P < 0.05). Scale bar = 1 cm. 3

4

Figure 4 Delayed germination of Pro35S:RCAR5 seeds under cold stress conditions is 5

regulated by downstream component HAB1, but not by ABI5. 6

A, B Germination rates of Pro35S:RCAR5, hab1, Pro35S:HAB1, 7

Pro35S:RCAR5/Pro35S:HAB1, and WT plants under cold stress conditions. Seeds were 8

plated on 0.5 MS medium and germinated in the dark at 4°C. For measuring germination 9

rates, the numbers of seeds with emerged radicles were counted at the indicated time points 10

(B). Representative images were taken at 20 DAI (A). 11

C, D Germination rates of Pro35S:RCAR5, abi5, Pro35S:RCAR5/abi5, and WT plants under 12

cold stress conditions. Representative images were taken at 18 DAI (C) and germination 13

rates were determined at 12 DAI and 14 DAI (D). Data represent mean ± SD of three 14

independent experiments, each evaluating 100 seeds. Different letters indicate significant 15

difference compared with WT (ANOVA; P < 0.05). Scale bar = 1 cm. 16

17

Figure 5 ABA-independent RCAR5 expression in Arabidopsis leaves in response to LT. 18

A RCAR expression in response to cold stress. Three-week-old Arabidopsis Col-0 plants 19

were exposed to 4°C and rosette leaves were harvested at the indicated time points. Actin8 20

was used as an internal control for normalization. COR15A was used as a positive control 21

for cold stress treatment. The expression level of each gene at 0 h was set to 1.0. 22

B RCAR5 expression pattern in ABA-deficient mutants aba1-6 and aba1-3 and ABA-23


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insensitive mutant abi5 in response to cold stress. Data represent mean ± SD of three 1

independent experiments. Asterisks indicate significant difference compared with non-2

treated control (Student’s t-test; P < 0.05). 3

4

Figure 6 Enhanced tolerance of Pro35S:RCAR5 plants to cold stress. 5

A, B Cold stress-induced dehydration of Pro35S:RCAR5 transgenic lines #1 and #2 and WT 6

plants. Three-week-old Arabidopsis plants were treated with cold stress (4°C) for 24 h and 7

representative images were taken (A). The fresh weights of each plant line (n = 30) were 8

measured at the indicated time points after cold stress (B). 9

C, D Leaf temperatures of Pro35S:RCAR5 plants in response to cold stress. Representative 10

thermographic images of Pro35S:RCAR5 and WT plants 4 h after cold stress treatment (C); 11

the mean leaf temperatures of the two largest leaves were measured using 20 plants of each 12

line (D). 13

E, F Stomatal apertures in Pro35S:RCAR5 transgenic lines and WT plants treated with cold 14

stress. Leaf peels harvested from 3-week-old plants of each line were incubated in chilled 15

stomatal opening solution (SOS) for 2 h at 4°C. Representative images were taken (E) and 16

the stomatal apertures were measured under the microscope (F). 17

G, H Freezing tolerance of Pro35S:RCAR5 and WT plants. Three-week-old seedlings of 18

each plant line were exposed to freezing temperatures as indicated. After recovery at 24°C 19

for 2 days, representative images were taken (G) and the survival rate of each line was 20

counted (H). 21

I Electrolyte leakage of Pro35S:RCAR5 and WT plants. For freezing treatment, 3-week-old 22

seedlings were exposed to −4°C for 1 h. All data represent mean ± SD of three independent 23


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experiments. Different letters indicate significant difference between WT and transgenic 1

plants (ANOVA; P < 0.05). 2

3

Figure 7 Stomatal closure and cold- and ABA-responsive gene expression of 4

Pro35S:RCAR5/aba1-6 plants under cold stress conditions. 5

A, B Leaf temperatures of Pro35S:RCAR5/aba1-6 plants in response to cold stress. 6

Representative thermographic images of Pro35S:RCAR5/aba1-6 and aba1-6 plants at 3 h 7

after cold stress treatment (A); the mean leaf temperatures of the two largest leaves were 8

measured using 24 plants of each line (B). 9

C Cold stress-induced dehydration of Pro35S:RCAR5/aba1-6 and aba1-6 plants. Three-10

week-old Arabidopsis plants were subjected to cold stress (4°C) for 6 h and representative 11

images were taken. 12

D Freezing tolerance of Pro35S:RCAR5/aba1-6 and aba1-6 plants. Three-week-old 13

seedlings of each plant line were exposed to freezing temperatures (−4°C) for 1 h and the 14

survival rate of each line was counted. 15

E Expression patterns of cold- and ABA-responsive genes in Pro35S:RCAR5/aba1-6 and 16

aba1-6 plants under cold stress conditions. 17

F Schematic representation of the functional role of RCAR5 in ABA-mediated seed 18

dormancy and ABA-independent stomatal closure under cold stress conditions. Arrows 19

indicate promotion actions; lines with end bar indicate inhibitory actions. 20

21

22


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1

Appendix Figures 2

Appendix Figure 1 Expression patterns of RCAR genes during seed germination and 3

enhanced ABA sensitivity of Pro35S:RCAR transgenic plants during seed germination 4

and seedling growth. (A) Relative expression levels of RCAR genes in dry seeds of 5

Arabidopsis ecotype Col-0 (upper) and Ler (bottom). Actin8 was used as an internal control 6

for normalization and the expression level of RCAR1 from each ecotype was set to 1.0. (B 7

and C) Relative expression levels of RCAR genes during germination. Data were obtained 8

from the Arabidopsis eFP Browser with ‘Germination’ (B) (Winter et al., 2007) and ‘Klepikova 9

Atlas’ (C) (Klepikova et al., 2016) as data sources in the Bio-Analytic Resource for Plant 10

Biology (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). H, seeds from the silque; S, 11

stratification; L, light. (D) Expression levels of RCAR genes in the leaves of Pro35S:RCAR 12

transgenic plants. Actin8 was used as an internal control for normalization and the 13

expression level of each RCAR gene in WT plants was set to 1.0. (E–G) Seedling 14

development of Pro35S:RCAR transgenic lines and WT plants in the presence of ABA. 15

Seeds of Pro35S:RCAR transgenic lines were germinated on 0.5 MS medium 16

supplemented with 0 μM or 0.5 μM ABA and grown at 24°C in the light. At 5 days after 17

incubation (DAI), cotyledon greening (F) and root length (G) were measured and 18

representative images were taken (E). Data represent mean ± SD of three independent 19

experiments. 20

21

Appendix Figure 2 ABA sensitivity of aba1-6, abi5, Pro35S:RCAR5/aba1-6, and 22

Pro35S:RCAR5/abi5 transgenic plants during seed germination and seedling growth. 23

(A) Expression levels of RCAR genes in the leaves of Pro35S:RCAR/aba1-6 transgenic 24

plants. Actin8 was used as an internal control for normalization and the expression level of 25


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RCAR5 in WT plants was set to 1.0. (B) Germination rates of Pro35S:RCAR, 1

Pro35S:RCAR/aba1-6, aba1-6, and WT plants on 0.5 MS medium supplemented with 0 μM 2

or 0.5 μM ABA. The numbers of seeds with emerged radicles were counted 3 days after 3

plating. (C–E) Seedling development of Pro35S:RCAR, Pro35S:RCAR/aba1-6, aba1-6, and 4

WT plants in the presence of ABA. Seeds of each plant line were germinated on 0.5 MS 5

medium supplemented with 0 μM, 0.5 μM, or 0.75 μM ABA and vertically grown at 24°C in 6

the light. At 7 days after incubation (DAI), root length (D) and cotyledon greening (E) were 7

measured and representative images were taken (C). (F) Expression levels of RCAR genes 8

in the leaves of Pro35S:RCAR/abi5 transgenic plants. Actin8 was used as an internal control 9

for normalization and expression level of RCAR5 in WT was set to 1.0. (G) Germination 10

rates of Pro35S:RCAR, Pro35S:RCAR/abi5, abi5, and WT plants on 0.5 MS medium 11

supplemented with 0 μM or 0.5 μM ABA. The numbers of seeds with emerged radicles were 12

counted 3 days after plating. (H–J) Seedling development of Pro35S:RCAR, 13

Pro35S:RCAR/abi5, abi5, and WT plants in the presence of ABA. Seeds of each plant line 14

were germinated on 0.5 MS medium supplemented with 0 μM or 0.75 μM ABA and 15

vertically grown at 24°C in the light. At 7 DAI, root length (I) and cotyledon greening (J) were 16

measured and representative images were taken (H). Data represent mean ± SD of three 17

independent experiments. Different letters indicate significant differences between three 18

independent experiments (ANOVA; P < 0.05). Scale bar = 1 cm. 19

20

Appendix Figure 3 Expression patterns of PP2C genes during seed germination and 21

after cold stress treatment, and reduced ABA sensitivity of Pro35S:HAB1 and 22

Pro35S:RCAR5/HAB1 transgenic plants during seed germination and seedling growth. 23

(A and B) Relative expression levels of PP2C genes during germination. Data were obtained 24

from the Arabidopsis eFP Browser with ‘Germination’ (A) (Winter et al., 2007) and ‘Klepikova 25


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Atlas’ (B) (Klepikova et al., 2016) as data sources in the Bio-Analytic Resource for Plant 1

Biology (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). H, seeds from the silque; S, 2

stratification; L, light. (C and D) Relative expression levels of PP2C genes in Arabidopsis 3

shoots and roots in response to cold stress. Data were obtained from the Arabidopsis eFP 4

Browser with ‘Abiotic stress’ (Winter et al., 2007) as the data source. (E) Expression levels of 5

RCAR genes in the leaves of Pro35S:HAB1 and Pro35S:RCAR/Pro35S:HAB1 transgenic 6

plants. Actin8 was used as an internal control for normalization. The expression levels of 7

RCAR5 or HAB1 genes in WT plants were set to 1.0. (F) Germination rates of Pro35S:RCAR, 8

Pro35S:HAB1, Pro35S:RCAR/Pro35S:HAB1, and WT plants on 0.5 MS medium 9

supplemented with 0 μM, 0.5 μM, or 1 μM ABA. The numbers of seeds with emerged 10

radicles were counted 3 days after plating. (G–I) Seedling development of Pro35S:RCAR, 11

Pro35S:HAB1, Pro35S:RCAR/Pro35S:HAB1, and WT plants in the presence of ABA. Seeds 12

of each plant line were germinated on 0.5 MS medium supplemented with 0 μM, 0.5 μM, or 13

0.75 μM ABA and vertically grown at 24°C in the light. At 7 DAI, root length (H) and 14

cotyledon greening (I) were measured and representative images were taken (G). Data 15

represent mean ± SD of three independent experiments. Different letters indicate significant 16

differences between three independent experiments (ANOVA; P < 0.05). Scale bar = 1 cm. 17

18

Appendix Figure 4 Sensitivity of hab1 and pp2ca mutant plants to cold stress. (A) 19

Physical interaction of RCAR5 with HAB1 and PP2CA in yeast. Interaction was indicated by 20

growth on selection medium (SC-Ade-His-Leu-Trp); growth on SC-Leu-Trp was used as a 21

control (right). (B) BiFC assay of interactions of RCAR5 with HAB1 and PP2CA. 22

RCAR5:VYNE was co-expressed with HAB1:CYCE or PP2CA:CYCE in the leaves of 23

tobacco. Scale bar = 20 mm. (C) Germination rates of Pro35S:RCAR5, hab1, pp2ca-1, and 24

WT plants. Seeds of these plants were plated on 0.5 MS medium and germinated in the 25


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dark at 4°C. (D) Vertical growth of Pro35S:RCAR5, hab1, pp2ca-1, and WT plants exposed 1

to ABA or LT. Seeds of each plant were plated on 0.5 MS medium supplemented with 0 μM 2

or 0.75 μM ABA and germinated under normal or cold stress conditions. Scale bar = 1 cm. 3

Representative images were taken at 8 DAI (for ABA) and 18 DAI (for LT). (E) qRT-PCR 4

analysis of HAB1 and PP2CA gene expression in Arabidopsis leaves in response to cold 5

stress (4°C). Actin8 was used as an internal control for normalization. The expression level 6

of each gene at 0 h was set to 1.0. (F–H) Leaf dehydration and leaf temperatures of 7

Pro35S:RCAR5, hab1, pp2ca-1, and WT plants in response to cold stress. Representative 8

thermographic images of Pro35S:RCAR5, hab1, pp2ca-1, and WT plants 24 h after 9

exposure to 4°C (F); the mean leaf temperatures of the two largest leaves were measured 10

using 20 plants of each line (G). Concomitantly, the fresh weights of each plant line were 11

measured (H). All data represent mean ± SD of three independent experiments. Different 12

letters indicate significant difference between WT and transgenic plants (ANOVA; P < 0.05). 13

14

Appendix Figure 5 Expression patterns of RCAR genes in Arabidopsis leaves after 15

cold stress treatment. (A) Relative expression levels of RCAR genes in the leaves of 16

Arabidopsis ecotype Col-0. Actin8 was used as an internal control for normalization, and the 17

expression level of RCAR1 was set to 1.0. (B) Relative expression levels of RCAR genes in 18

the leaves of aba1-6 and WT plants. (C–D) Relative expression levels of PP2C genes in 19

Arabidopsis shoots and roots in response to cold stress. Data were obtained from the 20

Arabidopsis eFP Browser with ‘Abiotic stress’ (Winter et al., 2007) as the data source in the 21

Bio-Analytic Resource for Plant Biology (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). (E) 22

Expression patterns of RCAR2, RCAR3, and RCAR5 in Arabidopsis leaves after dehydration 23

and ABA treatment. Relative expression levels of RCAR genes in Arabidopsis leaves after 24

ABA treatment and dehydration stress. Actin8 was used as an internal control for 25


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normalization. The expression level of each gene at 0 h was set to 1.0. (F and G) Enhanced 1

dehydration tolerance of Pro35S:RCAR5 transgenic plants. (F) Dehydration sensitivity of 2

Pro35S:RCAR5 and WT plants. Plants were subjected to dehydration stress by withholding 3

water for 12 days (n = 16); representative images were taken 3 days after rewatering. (G) 4

Water loss from leaves of WT and transgenic plants at various times after detachment of 5

leaves. Data represent mean ± standard error of three independent experiments (n = 20). 6

Different letters indicate significant difference between WT and transgenic plants (ANOVA; P 7

< 0.05). 8

9

Appendix Figure 6 Cold stress-induced dehydration of WT and Pro35S:RCAR2, 10

Pro35S:RCAR3, and Pro35S:RCAR5 transgenic plants. (A and B) Phenotypic response 11

of WT and transgenic lines to LT. Three-week-old Arabidopsis plants were exposed to 4°C 12

for 24 h and images were taken (A). Representative parts were magnified (B). Arrow heads 13

indicated wilted leaves. (C) Water loss from WT and Pro35S:RCAR2, Pro35S:RCAR3, and 14

Pro35S:RCAR5 plants after cold stress treatment. The fresh weights of each plant line were 15

measured 24 h after treatment. Data represent the mean ± SE of three independent 16

experiments, each evaluating 30 plants. Different letters indicate significant difference 17

between WT and transgenic plants (ANOVA; P < 0.05). (D and E) Thermal imaging analysis 18

of Pro35S:RCAR5 transgenic plants. Representative thermographic images of WT and 19

Pro35S:RCAR5 plants after LT treatment. Thermographic images were taken at different 20

time points after LT treatment (D), and the mean leaf temperatures of the two largest leaves 21

were measured using 20 plants of each line (E). Different letters indicate significant 22

differences (ANOVA; P < 0.05). 23

24

Appendix Figure 7 Strong upregulation of cold- and ABA-responsive genes in 25


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Pro35S:RCAR5 Pro35S:RCAR/Pro35S:HAB1, and Pro35S:RCAR/abi5 plants under 1

cold stress conditions. Two-week-old Pro35S:RCAR5 (A), Pro35S:RCAR/Pro35S:HAB1 2

(B), Pro35S:RCAR/abi5 (C) and WT plants were exposed to 4°C and shoots were harvested 3

at the indicated time points. Actin8 was used as an internal control for normalization. The 4

expression level of each gene at 0 h was set to 1.0. Data represent mean ± SD of three 5

independent experiments. Different letters indicate significant difference between WT and 6

transgenic plants (ANOVA; P < 0.05). 7

8

Appendix Table 1 Sequences of primers used in this study. 9


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