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

Cytological and histochemical analysis of siliques of oeWHY2, why2, and wild-type (WT) plants. A, Representative images of developing embryo in the seeds (7-week-old plant) are shown above the panels. Scale bars = 50 μm (as shown in the top left). B, Calculation of the embryo size in the silique of oeWHY2, why2, and wild-type plants. Twenty embryos per line were used. C, Observation of chloroplast ultrastructure. The red arrows indicate the starch granules. Scale bars = 250 nm. D, Calculation of the starch granules in the pericarp cells of oeWHY2, why2, and wild-type plants. Ten cells/silique and 5 chloroplasts/cell were used. Error bars represent the sd of triplicates. E, Starch staining of siliques including pericarp and seed in 9-week-old oeWHY2, why2, and wild-type plants. The red arrows indicate the seeds. Scale bars =10 mm. The siliques were separated to pericarp and seed to digitally extract for comparison. F and G, Starch content of siliques including pericarp (F) and seed (G) in 9-week-old oeWHY2, why2, and wild-type plants. Three plants, three siliques/plant, and 30 seeds/silique were used. Error bars represent the sd of triplicate reactions. Asterisks denote statistically significant differences from the wild type, calculated using Student’s t test: *P < 0.05; **P < 0.01; and ***P < 0.001.

The expression profile of select genes in the rosette leaf of oeWHY2, why2, and wild-type (WT) plants. A, heatmap showing the changes in expression of select genes (see Supplemental Fig. S4) in oeWHY2, why2, and wild type by RT-qPCR analysis. B, Confirmation of the expression of select genes in the two oeWHY2, two why2, and two comWHY2 lines, compared with wild type by RT-qPCR analysis. Error bars represent the sd of three biological replicates.

Detection of WHY2 triple localization. A, Schematic of a series of WHY2 deletion constructs. P2: WHY2 with deleted mitochondrion transit peptide fused to GFP. P4: WHY2 with deleted plastid and mitochondrial transit peptide fused to GFP. P6: full-length WHY2 fused to GFP. Scale bars = 50 μm. B, Positive control plasmids (COX-GFP, H2B-GFP, WHY1-GFP) expressed in onion epidermal cells by gene gun biolistic assay. C, Observation of P2, P4, and P6 subcellular localization in onion epidermal cells by gene gun biolistic assay. BF, bright field; Fluo, Fluorescence; DAPI, 4′,6-diamino-phenylindole staining the nucleus; scale bars = 50 μm. D, Observation of P2, P4, and P6 subcellular localization in Arabidopsis mesophyll cells by protoplast transit assay. The plasmids of COX-GFP, H2B-GFP, and WHY1-GFP were used as positive controls. Scale bars = 20 μm. E, Western blot detection of WHY2 expression in the mitochondria (Mito) fractions isolated from P6, nuclear fraction isolated from P4, and chloroplast (Chl) fraction isolated from P2 using an antibody against GFP. Anti-PSII was used as a chloroplast protein control, anti-H3 as a nuclear protein control, and anti-COXII as a mitochondrial protein control. Silver staining of the protein gel was used to indicate loading.

Detection of WHY2 triple localization during plant aging. A, Indication of three stages of wild-type (WT) plant development [4, 6, and 8 weeks (W)] by visible rosette leaf senescence. B, Indication of three stages of wild-type plant development (4, 6, and 8 weeks) and photosystem II fluorescence efficiency (Fv/Fm) and chlorophyll content. Error bars represent the sd of six biological replicates. C, WHY2 protein distribution among mitochondrion, chloroplast (Chl), and nucleus by immunodetection. Anti-PSII was used as a chloroplast protein control, anti-H3 as a nuclear protein control, and anti-VDAC1 (voltage-dependent anion channel 1) as a mitochondrial protein control. Silver staining of the protein gel was used to indicate loading.

WHY2 binds to the upstream region of NAD1 and ccb382 of the mitochondrial genome and promotes their expression. A, Diagram of the upstream regions of NAD1 and ccb382 genes. B, The sequences of the upstream regions of NAD1 and ccb382 genes. tel-cs, coding strand; tel-ncs, noncoding strand; tel-RNA, RNA sequence. C, EMSA. The tel-cs, tel-ncs, and tel-RNA fragments were used as the probe (labeled with biotin). Twentyfold excess competitor probe (without biotin) was added as a specificity control. The recombinant WHY2, expressed and isolated from E. coli, used in the reaction was detected by Western blot with anti-WHY2. D, Yeast one-hybrid assay results. The pNAD1, pccb382, and pATP9 fragments were inserted into the pHIS2 expression vector. The various dilutions of colonies on the selective medium showed the activation of expression by WHY2 of the HIS reporter gene driven by the indicated fragments in yeast. EV1, the plasmid GAD-WHY2 with the empty pHIS2; EV2, the empty plasmid GAD with promoter fragment fused HIS2 plasmid; they were cotransformed into the yeast strain AH109 as negative controls: P, positive, N, negative. E, Luciferase (LUC)/Renilase (REN) dual activation assay. Agrobacterium cells containing the vectors expressing WHY2-FLAG (ACTIN:WHY2-FLAG) and the Agrobacterium cells containing the vectors expressing fragments: LUC-REN were coinjected into Nicotiana benthamiana leaves. ATP9 promoter was used as a negative control. Shown are mean and SE of six biological replicates. Asterisks denote statistically significant differences from the empty vector, calculated using Student’s t test: *P < 0.05; **P < 0.01; and ***P < 0.001. F, Western blot detection of NAD1 and ccB382 protein levels in the oeWHY2 and why2 lines. COXII was used as a loading control.

WHY2 binds to upstream regions of JMT, SAG29, and SWEET11 and alters their expression. A, Diagram of the promoters of JMT, SAG29, SWEET11, and SWEET12 genes. B, Western blot detection of WHY2 protein in the rosette of 7-week-old oeWHY2, why2, and wild-type (WT) plants. C, Chromatin immunoprecipitation (ChIP) assay in rosette leaves of 7-week-old Arabidopsis oeWHY2, why2, and wild-type plants. Antibody against WHY2 peptide was used. TUB2 was used as a negative control. The fold enrichment of ChIP is relative to input. Data represent mean ± sd of five biological replicates. Asterisks denote statistically significant differences from the enrichment of TUB2, calculated using Student’s t test: (*P < 0.05; **P < 0.01, ***P < 0.001). D, LUC/REN dual activation assay, as above. Agrobacterium cells containing the vectors expressing WHY2-FLAG (ACTIN:WHY2-FLAG) or vectors expressing candidate promoter fragments plus GAL4: LUC-REN were coinjected into Nicotiana benthamiana leaves. The WRKY53 promoter was used as a negative control. E, Suc, Glc, and starch content in the leaf, pericarp, and seeds of 7-week-old oeWHY2, why2, and wild-type plants. Shown are mean ± se of six biological replicates. Asterisks denote statistically significant differences from the empty vector or wild type calculated using Student’s t test: *P < 0.05; **P < 0.01; and ***P < 0.001.

WHY2 alters carbon reallocation and leaf senescence in response to methyl jasmonate. A, Photograph of 4-week-old seedlings and iodine-stained rosettes of the oeWHY2, why2, comWHY2, and wild-type (WT) seedlings after methyl jasmonate (MeJA) treatment for 4 h. The degree of blue staining of the leaves (leaf 5, 6, 7) reflects the accumulation of starch. B, Determination of chlorophyll and starch content. The results were repeated in three independent experiments. Shown are the mean ± sd of three biological replicates. Asterisks denote statistically significant differences from the wild type calculated using Student’s t test: **P < 0.01. C, The expression levels of jasmonic acid-responsive genes and SWEET 11, 12, 15 genes in response to MeJA by RT-qPCR analysis. Shown are the mean ± sd of three biological replicates. Asterisks denote statistically significant differences from the wild type, calculated using Student’s t test: **P < 0.01 and ***P < 0.001.