Triple-localized WHIRLY2 Influences Leaf Senescence and Silique Development via Carbon Allocation.

Coordination of gene expression in mitochondria, plastids, and nucleus is critical for plant development and survival. Although WHIRLY2 (WHY2) is involved in mitochondrial genome repair and affects the DNA copy number of the mitochondrial genome, the detailed mechanism of action of the WHY2 protein is still elusive. In this study, we found that WHY2 was triple-localized among the mitochondria, plastids, and the nucleus during Arabidopsis (Arabidopsis thaliana) aging. Overexpressing WHY2 increased starch granule numbers in chloroplasts of pericarp cells, showing a partially dry, yellowing silique and early senescence leaves. Accordingly, WHY2 protein could directly activate the expression of jasmonic acid carboxyl methyltransferase and senescence associated gene 29 (SWEET15) gene expression and repress SWEET11 gene expression in the nucleus, leading to alteration of starch accumulation and transport in pericarp cells. In contrast, loss of WHY2 decreased starch and sugar content in pericarp cells but promoted starch accumulation in leaves and seeds. These phenotypes of WHY2-overexpressing plants were enhanced in response to methyl jasmonate. Our results suggest that WHY2 in plastids, mitochondria, and the nucleus plays a vital role in alteration of carbon reallocation from maternal tissue to filial tissue.


Introduction
There are three organelles in plant cells, which possess and maintain genetic 23 information: nucleus, plastids, and mitochondria. Coordination of gene 24 expression in these organelles is critical for plant development and survival 25 blue/black (Fig 2e). Starch was barely detectable in why2 pericarp cells, 152 whereas a dramatically stronger blue signal was observed in the oeWHY2 153 pericarp cells, relative to the wild type, indicative of reduced delivery of 154 sucrose to the seeds of oeWHY2 siliques (Fig 2e). Consistent with the optical 155 analysis of the starch staining, enzymatic quantitation of starch showed that 156 the pericarp cells of oeWHY2 siliques accumulated up to three times more 157 starch, compared to the wild type. However, the seeds of oeWHY2 158 accumulated up to five times less starch relative to the wild type (Fig 2f-g). The 159 accumulation of starch in the pericarp cells implicated a block in sugar release 160 from the pericarp cells as a key step in embryo development and seed filling. 161 Therefore, the abnormal development of silique caused by overexpressing 162 WHY2 may be related to the disturbance of starch output from chloroplasts of 163 maternal tissue pericarp cells or leaf cells. 164 decrease in chlorophyll content and fluorescence activity (Fv/Fm) of 237 photosystem II (Fig 5b). In contrast, WHY2 protein was relatively stable in 238 mitochondria during plant aging (Fig 5c), while WHY2 protein in plastids was 239 lower at week 4, increased at week 6, and slightly declined again at week 8, 240 when normalized to the loading control. Therefore, WHY2 is localized in all 241 three cellular compartments, and its location and distribution among cellular 242 compartments is developmentally dependent. 243 244 WHY2 directly activates NAD1 and ccb382 gene expression in the 245

mitochondrial genome 246
It is well documented that WHIRLY1 binds to the elicitor response element 247 (ERE) and AT-rich region (…AAAT…AAAT) repeat motifs and the telomere 248 repeat of downstream gene promoters (Desveaux et al., 2002;Yoo et al., 2007;249 Miao et al., 2013). Since WHIRLY proteins share the same ssDNA binding 250 domain, it is possible that WHY2 binds directly to similar fragments. To 251 address this possibility, we first screened for these sequence motifs in the 252 whole mitochondrial genome. Interestingly, there are several telomere repeats 253 existing in the upstream regions of NAD1 and ccb382 (Fig 6a). We chemically 254 synthesized the DNA fragments of upstream regions including four repeat 255 (4xTel-ncs), and RNA chain (4×Tel-RNA) (Fig 6b). The recombinant proteins 257 were expressed in E. coli and detected by western blot with an antibody 258 against a WHY2 peptide. Electrophoretic mobility shift assay (EMSA) was 259 used to detect the binding affinity of WHY2 to upstream regions of NAD1 and 260 ccb382 genes. The probes were labeled with 32 P and incubated with WHY2. 261 The unlabeled fragment was used as a competitive probe. As more 262 competitive probe was added, the binding signal became weaker (Fig 6c). 263 These results indicated that WHY2 protein bound to 4xTel-ncs and 4xTel-RNA 264 fragments of the upstream regions of the NAD1 and ccb382 genes of the 265 mitochondrial genome in vitro, but the 4xTel-cs did not show good competition 266 and had weaker binding. A similar result was obtained for WHY1 binding to 267 Pccb382:HIS2 were activated, as indicated by colony growth on selective 277 medium (Fig 6d). In contrast, no growth was observed with PATP9:HIS2 (Fig  278   6d). 279 The binding of WHY2 to the upstream regions of NAD1, ccb382, and ATP9 280 was examined using the transient dual-luciferase assay system in Nicotiana 281 benthamiana leaves (Hellens et al., 2005). The promoter sequences of NAD1, 282 ccb382, and ATP9 were cloned into dual-luciferase vectors. The WHY2 coding 283 sequence, fused to FLAG-tag and under the control of the Arabidopsis ACTIN1 284 promoter (ACTIN:WHY2-HA), was co-infiltrated with reporter vector containing 285 the above putative promoter sequence of the tested genes fused to the 286 luciferase (LUC) and rennilase (REN) reporter (Hellens et al., 2005). We then 287 measured the LUC and REN luminescence ratio (LUC/REN ratio) in infiltrated 288 leaves. To assess any basal activation or repression of putative promoters, a 289 mini-GAL4 promoter vector was used in each co-infiltration experiment as a 290 control; ATP9 promoter was used as a negative control. The results showed 291 high LUC/REN ratio with the promoters of NAD1 and ccb382 in the presence 292 of WHY2 (Fig 6e). Therefore, WHY2 directly activates NAD1 and ccb382 293 expression. Surprisingly, NAD1 protein substantially accumulated in the why2 294 plants, while ccb382 protein maintained the same level or slightly increased in 295 the why2 plants (Fig 6f). Additionally, NAD1 dramatically declined upon 296 overexpression of WHY2 in the why2 background (oeWHY2/why2), as 297 assessed by immunodetection (Fig 6f). We hypothesize that NAD1 and 298 ccb382 protein levels were affected by WHY2 at the posttranscriptional level. 299 Taken together, our results show that WHY2 functions as a DNA/RNA-binding 300 protein to activate NAD1 and ccb382 gene transcription. However, WHY2 301 suppresses NAD1 and ccb382 protein accumulation in the mitochondrion. 302 303 Nuclear WHY2 directly binds to the promoter region of JMT, SAG29, and 304

SWEET11 genes and alters their expression level and carbon allocation 305
As shown above, WHY2 is also localized in the nucleus. As WHY2 is an 306 ssDNA-binding protein in the nucleus, it is possible that WHY2 directly binds to 307 plants, compared to the samples of the why2 line or wild type (Fig 7b). A 318 strong binding signal of JMT, SAG29, and SWEET11 was detected in the 319 samples from the oeWHY2 line, and a modest signal was detected in the wild 320 type samples (Fig 7c). These results suggested that WHY2 could directly bind 321 to these regions of JMT, SAG29, and SWEET11, but not MYB113 and 322 SWEET12. The promoter region of tubulin beta chain 2 (TUB2) (-246 to 323 -362bp), which was used as negative control, was not enriched in the ChIP 324 samples (Fig 7d).  (Fig 7d). Surprisingly, a 1000-fold increase was observed with the JMT 336 promoter, while no change was observed with SWEET12 and WRKY53. These 337 results suggest that WHY2 directly binds to the promoter regions of JMT, 338 while repressing SWEET11 gene expression, consistent with the gene 340 expression profile detected by RT-qPCR (Fig 3). 341 342 SAG29 was first identified as a jasmonic acid (JA)-mediated senescence 343 marker gene, highly expressed in senescent leaves (Seo et al., 2001;Qi et al., 344 2015). It is a membrane protein that is able to transport sucrose, which led to it 345 later being renamed SWEET15. SWEETs were prime candidates to play roles 346 in sugar secretion from maternal tissues (leaf, pericarp, seed coat) during seed 347 development (Chen et al., 2014). Since WHY2 activated SAG29 (SWEET15) 348 gene expression and repressed SWEET11 gene expression, WHY2 should 349 affect sucrose transport from chloroplast of leaf and pericarp to other 350 compartments or tissues. To test this, we measured the starch accumulation 351 and sugar (sucrose and glucose) content of leaf, pericarp, and seed of 352 7-week-old oeWHY2, why2, and wild-type plants. Interestingly, the starch 353 content and sugar content were significantly increased in the pericarp cells of 354 siliques but decreased in the seeds of the oeWHY2 line. In contrast, the starch 355 content and sugar (sucrose and glucose) content declined five-fold in the 356 pericarp cells of the why2 line. While the starch content significantly increased 357 in the seeds of the why2 plants, the sugar content was not changed in the 358 seeds of the why2 line, compared to the wild type. The starch accumulation 359 and sugar content in stems did not change significantly in response to varying 360 levels of WHY2. However, the content of starch and sugar in leaves decreased 361 significantly in the oeWHY2 plants, and increased in the why2 plants (Fig 7e). 362 Therefore, it seems that WHY2 affects sucrose transport from leaf to pericarp 363 cells via SWEET15 and from pericarp cells to seeds via SWEET11. 364 365 WHY2 levels affect JMT and SAG29 levels and starch content in the 366 seedling in response to JA 367 In addition to SAG29 and SWEET11, WHY2 could activate JMT expression 368 and repress JAS and TAT3 gene expression (Fig 3). Therefore, WHY2 may be 369 involved in the JA responsive signal pathway. To test this possibility, we treated 370 4-week-old oeWHY2, why2, and WT seedlings with 100 mM MeJA for 1, 2, 3, 371 and 4 h (Supplemental Fig S5), and observed the senescence phenotype of 372 seedlings and the starch content of leaves (in the fifth, sixth, and seventh 373 leaves). Iodine staining of seedlings showed that a blue signal was enhanced 374 in why2 seedlings and declined in oeWHY2 seedlings, compared to wild type, 375 after MeJA treatment for 4 h, demonstrating an accelerated early senescence 376 phenotype (Fig 8a). Starch content and chlorophyll content declined 377 significantly in the leaves of the oeWHY2 line in response to MeJA (Fig 8b). 378 Surprisingly, the gene expression levels of WHY2, JMT, and SWEET15 were 379 largely increased in the oeWHY2 and wild-type plants, but that of SWEET11 380 was significantly decreased after MeJA treatment (Fig 8c). This result suggests 381 that WHY2 accelerates sucrose transport out of leaves, but inhibits sucrose 382 transport from pericarp cells to seeds in response to MeJA. Thus, sugar 383 starvation might cause early leaf senescence and at the same time delay 384 (embryo) seed development. 385 386

Discussion 387
Effective degradation and remobilization of macromolecules is important for 388 senescence and successful reproduction (Gepstein et al., 2003). In this study, 389 we found that WHY2, a protein triple-localized among mitochondrion, plastids, 390 and the nucleus, has altered localization during plant aging. It works as a 391 WHY2 enhanced starch accumulation in chloroplasts of pericarp cells, leading 394 to withered, yellowed, and premature senescence phenotypes of leaves and 395

siliques. 396
One of the WHIRLY family proteins, WHIRLY1, has dual locations and 397 functions (Krause et al., 2005;Grabowski et al., 2008;Ren et al., 2017). It has 398 been reported, using an in vitro import assay, that the WHY2 protein could be 399 imported into mitochondria and plastids (Krause et al., 2005). This study 400 further addresses the subcellular localization of WHY2, and shows that it is 401 localized in three compartments (mitochondria, plastids, and the nucleus) 402 during plant aging (Fig 5), with nuclear localization mainly in senescent leaf 403 cells. In mitochondria, WHY2 could directly bind to the 4x telomere repeat 404 DNA/RNA fragments of the upstream regions of NAD1 and ccb382 genes, and 405 activate NAD1 and ccb382 gene transcription, but suppress NAD1 protein 406 accumulation (Fig 6). NAD1 is known to be a critical component of  (Law et al., 2015). It is hypothesized that mtWHY2 takes part in maintaining 437 the respiratory electron transport chain by controlling NAD1 and ccb382 levels 438 The chlorophyll content of leaf 7 was measured according to the method 527 described previously (Porra et al., 1989). At least 12 individual plants were 528 used for analysis and the mean chlorophyll content was calculated. 529

Plasmid Construction and Plant Transformation 530
A series of WHY2 CDS constructs including the full-length (714bp) mtWHY2, 531 the deleted mitochondrial transit peptide (627bp) ptWHY2, and the deleted 532 mitochondrial and plastid transit peptide (576bp) nWHY2 were amplified with 533 primers adding CACC at the 5' end and a FLAG tag at the 3' end 534 (Supplemental Table S1), and cloned in a pENTR-TOPO vector using the 535 Gateway system (Invitrogen). The sequence of WHY2 was verified by 536 sequencing. Then WHY2 was transferred into the destination vector pB2WG7 537 by recombination using the Gateway system (Invitrogen). A positive clone of 538 Agrobacterium tumefaciens GV3101 was obtained and transformed into the  S1). 547

Northern blotting 548
10 μg of total RNA was run on a denaturing agarose gel in the presence of 549 formaldehyde, and then transferred to a membrane. The membrane was 550 pre-hybridized, hybridized with probe of the WHY2 CDS labeled with 32 P, then 551 the results were obtained by X-ray film exposure using a Phosphor Imager, as 552 described previously (Miao and Zentgraf, 2007). 553

Western blotting 554
The procedure of protein isolation and immunodetection was described

Phenotype observation and documentation 562
For phenotype observation, thirty siliques of three plants were used. Silique 563 size (width and length) was measured using a measuring stick. Seed weight 564 was calculated as the weight per 100 seeds. Siliques were opened on a 565 microscope slide right after collection. Thirty seeds were mounted in water, 566 and gentle pressure was applied to the cover slip on the seeds to release the 567 www.plantphysiol.org on September 17, 2020 -Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
embryos. Afterwards, non-damaged embryos were documented under a Leica 568 stereomicroscope. Twenty embryos per line were observed, and the area of 569 embryos were calculated using a width x length measurement. 570

Tissue section and ultrastructural observation 571
The silique of a 7-week-old plant was fixed in 100 ml FAA( containing 38% (v/v) 572 Formaldehyde, glacial Acetic acid, and 70% (v/v) Alcohol (0.5 : 0.5 : 9)) for 12 573 h, followed by alcohol gradient dehydration, clearing, wax immersion, paraffin 574 embedding, and cutting with a slice thickness 5-8 µm, and organizing the 575 structure. Photographs were taken using a Leica microscope. 576 For transmission electron microscopy, the fresh fruit were placed in 2.5% (v/v) 577 glutaraldehyde for 2 h, then rinsed with 1% (v/v) osmium tetroxide. After 1 h of 578 fixation, the specimen was rinsed with phosphate buffer and dehydrated with 579 acetone gradient. After embedding with epoxy resin, the specimen was 580 prepared by hand and then ultrathin sections were prepared. The ultrathin 581 sections were incubated with 1% (w/v) uranyl acetate and lead citrate and 582 examined with an electron microscope (TECNAI G2 20; FEI) at an 583 accelerating voltage of 120 kV.  Table S1. 594 regions in the downstream target genes, JMT and SWEET11/15, we used 609 four-week-old rosettes from oeWHY2-1, wild-type, and why2-1 plants. The 610 cross-linked DNA fragments ranging from 200 to 1000 bp in length were 611 immunoprecipitated by an antibody against the WHY2 peptide (Faan, 612 Hangzhou, China), whose specificity is shown in Supplemental Fig S2. The 613 enrichment of the selected promoter regions of both genes was determined by 614 comparing the amounts in the precipitated and non-precipitated (input) DNA 615 samples, by qPCR using designed region-specific primers (Supplemental 616 Table S1). The same quantification in the why2 mutant line served as a control 617 for the respective overexpressing line and wild type, and was used for  chloroplast fraction isolated from P2 using an antibody against GFP. Anti-PSII 773 was used as a chloroplast protein control, anti-H3 as a nuclear protein control, 774 and anti-COXII as a mitochondrial protein control. Silver staining of the protein 775 gel was used to indicate loading. 776 immunoprecipitation (ChIP) assay in rosette leaves of 7-week-old Arabidopsis 820 oeWHY2, why2, and WT plants. Antibody against WHY2 peptide was used. 821 TUB2 was used as a negative control. The fold enrichment of ChIP is relative 822 to input. Data represent mean ± SD of five biological replicates. Asterisks 823 denote statistically significant differences from the enrichment of TUB2, 824 calculated using Student's t test: (*, P < 0.05; **, P < 0.01, ***, P < 0.001); (d). 825 Luciferase (LUC)/Renilase (REN) dual activation assay, as above. 826 Agrobacterium cells containing the vectors expressing WHY2-FLAG 827 (ACTIN:WHY2-FLAG) or vectors expressing candidate promoter fragments 828 plus GAL4: LUC-REN were co-injected into Nicotiana benthamiana leaves. 829 The WRKY53 promoter was used as a negative control. (e). Sucrose, glucose, 830 and starch content in the leaf, pericarp, and seeds of 7-week-old oeWHY2, (leaf 5, 6, 7) reflects the accumulation of starch. (b). Determination of 840 chlorophyll and starch content. The results were repeated in three independent 841 experiments. Shown are the mean ± SD of three biological replicates. 842 Asterisks denote statistically significant differences from the WT calculated 843 using Student's t test: **, P < 0.01; (c). The expression levels of jasmonic 844 acid-responsive genes and SWEET 11, 12, 15 genes in response to MeJA by 845 RT-qPCR analysis. Shown are the mean ± SD of three biological replicates. 846 Asterisks denote statistically significant differences from the WT, calculated 847 using Student's t test: **, P < 0.01; and ***, P< 0.001. Overexpressing WHY2 in leaf cells enhances JMT and SWEET15 expression. 860