Walnut phosphatase 2A proteins interact with basic leucine zipper protein JrVIP1 to regulate osmotic stress response <i>via</i> calcium signaling

Yi He; Shuwen Chen; Chenhao Li; Shen Yang; Zhongyu Sun; Siyu Hou; Zhenggang Xu; Guiyan Yang; Yi He; Shuwen Chen; Chenhao Li; Shen Yang; Zhongyu Sun; Siyu Hou; Zhenggang Xu; Guiyan Yang

doi:10.48130/forres-0024-0012

Figures (7) Tables (2)

Figure 1.
Distribution of the JrPP2As on chromosomes of the J. regia genome. The chromosome number is shown on the top side of each chromosome.
Figure 2.
Phylogenetic relationship of PP2A proteins from J. regia, A. thaliana and H. brasiliensis. A, C, B55, B', B" means five sub-family of PP2As, respectively, which are displayed in different colors. A total of 15 walnut PP2As are represented by yellow five-pointed stars, 25 Arabidopsis PP2As are represented by red circles, 36 H. brasiliensis PP2As are represented by blue triangles.
Figure 3.
Gene structure of walnut PP2As. (a) Exon-intron distribution map. The vertical phylogenetic tree and gene structure of JrPP2As was constructed by GSDS online software. Yellow boxes indicate exons; blue boxes indicate upstream or downstream; black lines indicate introns. (b) Conserved motif analysis: 20 separate patterns were identified with the MEME suite and each pattern was depicted with different colors. (c) Distribution of conserved domains.
Figure 4.
QRT-PCR analysis of the expression of JrPP2A genes under drought, NaCl, CaCl_2,and ABA treatments. The relative expression level is expressed as relative to the internal reference gene and at 0 h. Error bars represent the SD (n = 3). Lowercase letters indicate significant differences among different JrPP2A genes under each treatment according to the Student's t-test (p < 0.05). (a) PEG₆₀₀₀ stress. (b) NaCl stress. (c) CaCl₂ treatment. (d) ABA treatment.
Figure 5.
Osmotic stress response function of JrPP2As. WT, wild type. JrPP2A07, 09, 14, the transgenic lines overexpression of JrPP2A07, JrPP2A09, JrPP2A14. Fourty two-day old seedlings were treated with PEG₆₀₀₀, PEG₆₀₀₀+ CaCl₂, PEG₆₀₀₀+ ABA, NaCl, NaCl + CaCl₂, NaCl + ABA for 5 d. Control was normally watered. The significant differences among WT, JrPP2A07, JrPP2A09, and JrPP2A14 were marked with lowercase (p < 0.05). (a) NBT staining. (b) Total ROS content. (c) Total Ca content.
Figure 6.
Interaction analysis of JrVIP1 and JrPP2A proteins using yeast two-hybrid (Y2H) and in vitro pull-down assay. AD+BD, AD+BD-JrVIP1, AD-JrVIP1+BD, negative control. The DDO plate was used as positive control for growth. (a) JrVIP1 was used as the bait. (b) JrVIP1 was used as the pray. (c) An in vitro pull-down assay demonstrates the interaction between JrVIP1 with JrPP2A07, JrPP2A09, JrPP2A14. JrPP2A-His protein was incubated with immobilized GST or GST-JrVIP1 protein, and immunoprecipitated fractions were detected by anti-His antibody. The assay was performed three times with the same result.
Figure 7.
The JrVIP1 and JrPP2As mediated osmotic stress responsive pathway in walnut trees.

Gene names	Type	Transcriptome No.	Gene Bank accession No.	Gene ID	Chromosome	ORF length (bp)	Number of amino acids	Molecular weight (kDa)	pI
JrPP2A01	B'	comp30409_c0	XM_018967286.2	LOC108992675	ch1	1,452	483	55.13	8.37
JrPP2A02	B''	comp28147_c0	XM_018971752.2	LOC108996025	ch1	1,254	417	49.44	4.8
JrPP2A03	B55	comp26329_c0	XM_018982199.2	LOC109003870	ch2	1,506	501	56.72	6
JrPP2A04	B'	comp20037_c0	XM_018993415.2	LOC109011996	ch3	1,503	500	57.52	6.24
JrPP2A05	C	comp26715_c0	XM_018952439.2	LOC108981323	ch5	681	226	25.7	4.94
JrPP2A06	B'	comp24655_c0	XM_018979195.2	LOC109001785	ch8	1,503	500	56.94	7.61
JrPP2A07	C	comp32898_c0	XM_018969187.2	LOC108994090	ch9	918	305	34.78	5.24
JrPP2A08	B'	comp25187_c0	XM_018958784.2	LOC108986228	ch10	1,575	524	59.87	7.97
JrPP2A09	B''	comp9850_c0	XM_018985083.2	LOC109005962	ch12	1,431	476	54.74	4.93
JrPP2A10	A	comp28348_c0	XM_035685846.1	LOC109013629	ch15	1,764	587	65.61	4.88
JrPP2A11	B55	comp28413_c1	XM_018989098.2	LOC109008856	ch15	918	305	34.06	5.16
JrPP2A12	B55	comp28413_c1	XM_018989097.2	LOC109008856	ch15	651	216	24.15	6.21
JrPP2A13	C	comp27670_c1	XM_018980847.2	LOC109002920	ch15	912	303	34.84	4.88
JrPP2A14	C	comp23892_c0	XM_018963562.2	LOC108989812	ch16	921	306	35.01	4.83
JrPP2A15	B'	comp13497_c0	XM_018951013.2	LOC108980163	ch16	1,530	509	58.52	7.18

Table 1.

Sequence characteristics of 15 JrPP2As.

Motif	Width	Motif consensus
1	41	TIVYGFYDETERHNGIAELLEIFGSIIDGFALPLKEEHKIF
2	50	KVAKRYIDHSFVLRLLDLFDSEDPREREYLKTILHRIYGKFMVHRPFIRK
3	50	HKPKSIGLYHQQLSYCITQFVEKDPKLADTVIRGLLKYWPVTNSQKEVMF
4	50	PAEFQRCMVPLFRQIGCCLNSSHFQVAERALFLWNNDHIVNLIAQNRNVI
5	50	NVQPVKSPVTICGDIHGQFHDLIELFRIGGNCPDTNYLFMGDYVDRGYYS
6	50	PIIFPALEKNARSHWNQAVQNLTLNVRKIFSEMDPELFEECQRQFQEDEA
7	48	DIKRQTLIELVDFVASGSGKFTETAIQEMIKMVSVNLFRVLPPKPREN
8	41	CLHGGLSPSIETLDNIRVIDRIQEVPHEGPMCDLLWSDPDD
9	29	EPSFDPAWPHLQJVYELLLRFVSSSETDA
10	37	VEALPAFKDVPNSEKQNLFISKLNLCCVVFDFSDPTK
11	29	ETFTLLLALKVRYPDRITJLRGNHESRQI
12	41	WGVSPRGAGYLFGGDVVSQFNHTNNLDLICRAHQLVMEGYK
13	27	WFQDKGIVTVWSAPNYCYRCGNVAAIL
14	38	SHADLDRQIEQLKECKPLPEAEVKVLCDKAKEILVEES
15	50	AHAHDFNINSISNNSDGETFISADDLRINLWNLEISNQCFNIIDMKPANM
16	50	YKTEFQSHEPEFDYLKSLEIEEKINKIRWCQTQNGALFLLSSNDKTIKFW
17	50	MDSGPVATFKVHENLRPKLCELYENDSIFDKFECCJSGDGJHFATGSYSN
18	38	EVITSAEFHPIHCNLLAYSSSRGFIRLIDMRCSALCDQ
19	17	TMIKQILSKLPRKPSKS
20	50	PLEWKFSQVFGERPAGEEVQEVDIISAIEFDKSGDHLAVGDRGGRVVJFE

Table 2.

Motif sequences identified by the MEME tool.