Metabolomics reveals early pregnancy biomarkers in sows: a non-invasive diagnostic approach.

In an effort to enhance reproductive management and reduce non-productive periods in swine breeding, this study presents a novel, non-invasive metabolomics approach for the identification of early pregnancy biomarkers in sows. Utilizing an untargeted metabolomics approach with mass spectrometry analysis, we examined saliva samples from pregnant (n = 6) and non-pregnant control sows (n = 6, artificially inseminated with non-viable sperm). Our analysis revealed 286 differentially expressed metabolites, with 152 being up-regulated and 134 down-regulated in the pregnant group. Among these, three metabolites, namely Hyodeoxycholic acid, 2'-deoxyguanosine, and Thymidine, emerged as potential early pregnancy biomarkers. These biomarkers were further evaluated using targeted LC-MS/MS quantification and qualification, accompanied by ROC curve analysis. The study confirmed Hyodeoxycholic acid and 2'-deoxyguanosine as promising biomarkers for early pregnancy detection, offering potential for future implementation in swine production environments. This research establishes a robust theoretical foundation for the development of innovative molecular diagnostic techniques and explores new avenues for molecular genetic breeding and non-invasive diagnostics, ultimately enhancing fertility and productivity in sow herds.

NtMYB12 requires for competition between flavonol and (pro)anthocyanin biosynthesis in Narcissus tazetta tepals.

The color of flowers is one of the main characteristics adopted for plants to attract pollinators to ensure the reproductive success of the plant, they are also important in their ornamental appeal in Narcissus plant. In this study, we identified a NtMYB12 locus encoding an R2R3-MYB transcription factor. Comparative transcriptome analysis of loss- and gain- of NtMYB12 tissue relative to wild-type narcissus showed NtMYB12 was mainly involved in flavonol and phenylpropanoid metabolic pathways. Biochemical evidences of dual-luciferase activity and chromatin immunoprecipitation assay supported that MYB12 directly bound to promoters of NtFLS, NtLAR, and NtDFR that were cloned by genome walking assay, and activated NtFLS and NtLAR expression but repressed NtDFR expression. More interestingly, NtMYB12 can interact with NtbHLH1 and NtWD40-1 proteins via R3 domain that were selected by transcriptome-based WGCNA and confirmed by yeast two hybrid, bimolecular fluorescence complementation and coimmunoprecipitation assay. Interaction of NtMYB12 with NtbHLH1 and NtWD40-1 forming MYB-bHLH-WD40 triplex specially activated NtDFR and NtANS expression and promoted (pro)anthocyanin accumulation, while NtMYB12 alone activated NtFLS and NtLAR expression and accumulated flavonols, but repressed NtDFR expression. These results indicated that NtMYB12 alone or NtMYB12-bHLH1-WD40-1 triplex requires for competition of metabolism fluxes between flavonol and (pro)anthocyanin biosynthesis. NtMYB12 dually functions on flavonol and proanthocyanin biogenesis via physically binding to NtFLS and NtLAR promoter activating their expression and on (pro)anthocyanin biosynthesis via NtMYB12-NtWD40-NtbHLH (MBW) triplex activating NtDFR and NtANS expression. Requirement of NtMYB12 alone or MBW complex for the competition between flavonol and anthocyanin biosynthesis results in narcissus colorized petal traits.

NtMYB1 and NtNCED1/2 control abscisic acid biosynthesis and tepal senescence in Chinese narcissus (Narcissus tazetta).

Chinese narcissus (Narcissus tazetta var. chinensis cv. 'Jinzhanyintai') is one of the 10 most famous traditional flowers of China, having a beautiful and highly ornamental flower with a rich fragrance. However, the flower longevity affects its commercial appeal. While petal senescence in Narcissus is ethylene-independent and abscisic acid-dependent, the regulatory mechanism has yet to be determined. In this study, we identified a R2R3-MYB gene (NtMYB1) from Narcissus tazetta and generated oeNtMYB1 and Ntmyb1 RNA interference mutants in Narcissus as well as an oeNtMYB1 construct in Arabidopsis. Overexpressing NtMYB1 in Narcissus or Arabidopsis led to premature leaf yellowing, an elevated level of total carotenoid, a reduced level of chlorophyll b, and a decrease in photosystem II fluorescence (Fv/Fm). A dual-luciferase assay and chromatin immunoprecipitation-quantitative PCR revealed that NtMYB1 directly binds to the promoter of NtNCED1 or NtNCED2 and activates NtNCED1/2 gene expression both in vitro and in vivo. Moreover, overexpressing NtMYB1 accelerated abscisic acid biosynthesis, up-regulated the content of zeatin and abscisic acid, and down-regulated the level of ß-carotene and gibberellin A1, leading to petal senescence and leaf yellowing in Narcissus. This study revealed a regulatory process that is fundamentally different between non-photosynthetic organs and leaves.

Retraction Notice to: UPL5 modulates WHY2 protein distribution in a Kub-site dependent ubiquitination in response to [Ca2+]cyt-induced leaf senescence.

[This retracts the article DOI: 10.1016/j.isci.2023.106216.].

UPL5 modulates WHY2 protein distribution in a Kub-site dependent ubiquitination in response to [Ca2+]cyt-induced leaf senescence.

The translocation of proteins between various compartments of cells is the simplest and most direct way of an/retrograde communication. However, the mechanism of protein trafficking is far understood. In this study, we showed that the alteration of WHY2 protein abundance in various compartments of cells was dependent on a HECT-type ubiquitin E3 ligase UPL5 interacting with WHY2 in the cytoplasm, plastid, and nucleus, as well as mitochondrion to selectively ubiquitinate various Kub-sites (Kub 45 and Kub 227) of WHY2. Plastid genome stability can be maintained by the UPL5-WHY2 module, accompany by the alteration of photosystem activity and senescence-associated gene expression. In addition, the specificity of UPL5 ubiquitinating various Kub-sites of WHY2 was responded to cold or CaCl2 stress, in a dose [Ca2+]cyt-dependent manner. This demonstrates the integration of the UPL5 ubiquitination with the regulation of WHY2 distribution and retrograde communication between organelle and nuclear events of leaf senescence.

Identification and expression profiles of xylogen-like arabinogalactan protein (XYLP) gene family in Phyllostachys edulis in different developmental tissues and under various abiotic stresses.

Xylogen-like arabinogalactan protein (XYLP) is an atypical lipid transport protein. In this study, 23 Phyllostachys edulis XYLPs were identified, and their proteins contain characteristic structures of AGP and nsLTP domain. All PeXYLPs can be divided into four clades, and their genes were unevenly distributed on 11 chromosome scaffolds. Collinear analysis revealed that segmental duplication was the main driver for PeXYLP family expansion. The cis-acting elements presented in the promoter are involved in various regulations of PeXYLPs expression. G.O. annotation revealed that PeXYLPs are mainly interested in lipid transport and synthesis and primarily function at the plasma membrane. Transcriptome analysis revealed that PeXYLPs were spatiotemporally expressed and displayed significant variability during various tissue development. Besides that, some PeXYLPs also respond to multiple phytohormones and abiotic stresses. By semi-quantitative RT-PCR, the response of some PeXYLPs to MeJA was confirmed, and the proteins were shown to localize to the plasma membrane mainly. WGCNA in defined regions of fast-growing bamboo shoots revealed that 5 PeXYLPs in 4 gene co-expression modules showed a positive module-trait relationship with three fast-growing regions. This systematic analysis of the PeXYLP family will provide a foundation for further insight into the functions of individual PeXYLP in a specific tissue or organ development, phytohormone perception, and stress responses in the future.

Genes, Structural, and Biochemical Characterization of Four Chlorophyllases from Solanum lycopersicum.

Recent studies have confirmed that chlorophyllase (CLH), a long-found chlorophyll (Chl) dephytylation enzyme for initiating Chl catabolism, has no function in leaf senescence-related Chl breakdown. Yet, CLH is considered to be involved in fruit degreening and responds to external and hormonal stimuli. The purpose of this work was to elucidate in detail the biochemical, structural properties, and gene expression of four CLHs from the Solanum lycopersicum genome so as to understand the roles of Solanum lycopersicum chlorophyllases (SlCLHs). SlCLH1/4 were the predominantly expressed CLH genes during leaf and fruit development/ripening stages, and SlCLH1 in mature green fruit was modulated by light. SlCLH1/2/3/4 contained a highly conserved GHSXG lipase motif and a Ser-Asp-His catalytic triad. We identified Ser159, Asp226, and His258 as the essential catalytic triad by site-directed mutagenesis in recombinant SlCLH1. Kinetic analysis of the recombinant enzymes revealed that SlCLH1 had high hydrolysis activities against Chl a, Chl b, and pheophytin a (Phein a), but preferred Chl a and Chl b over Phein a; SlCLH2/3 only showed very low activity to Chl a and Chl b, while SlCLH4 showed no Chl dephytylation activity. The recombinant SlCLH1/2/3 had different pH stability and temperature optimum. Removal of the predicted N-terminal processing peptide caused a partial loss of activity in recombinant SlCLH1/2 but did not compromise SlCLH3 activity. These different characteristics among SlCLHs imply that they may have different physiological functions in tomato.

MicroRNA840 (MIR840) accelerates leaf senescence by targeting the overlapping 3'UTRs of PPR and WHIRLY3 in Arabidopsis thaliana.

MicroRNAs negatively regulate gene expression by promoting target mRNA cleavage and/or impairing its translation, thereby playing a crucial role in plant development and environmental stress responses. In Arabidopsis, the MIR840 gene is located within the overlapping 3'UTR of the PPR and WHIRLY3 (WHY3) genes, both being predicted targets of miR840* and miR840, the short maturation products of MIR840. Gain- and loss-of-function of MIR840 in Arabidopsis resulted in opposite senescence phenotypes. The highest expression levels of the MIR840 precursor transcript pre-miR840 were observed at senescence initiation, and pre-miR840 expression is significantly correlated with a reduction in PPR, but not WHY3, transcript levels. Although a reduction of transcript level of PPR, but not WHY3 transcript levels were not significantly affected by MIR840 overexpression, its protein levels were strongly reduced. Mutating the cleavage sites or replacing the target sequences abolishes the miR840*/miR840-mediated degradation of PPR transcripts and accumulation of WHY3 protein. In support for this, concurrent knockdown of both PPR and WHY3 in wild-type plants resulted in a senescence phenotype resembling that of the MIR840-overexpressing plant. This indicates that both PRR and WHY3 are targets in the MIR840-mediated senescence pathway. Moreover, single knockout mutants of PPR and WHY3 show a convergent upregulated subset of senescence-associated genes, which are also found among those induced by MIR840 overexpression. Our data provide evidence for a regulatory role of MIR840 in plant senescence.

Transcriptome-Based WGCNA Analysis Reveals Regulated Metabolite Fluxes between Floral Color and Scent in Narcissus tazetta Flower.

A link between the scent and color of Narcissus tazetta flowers can be anticipated due to their biochemical origin, as well as their similar biological role. Despite the obvious aesthetic and ecological significance of these colorful and fragrant components of the flowers and the molecular profiles of their pigments, fragrant formation has addressed in some cases. However, the regulatory mechanism of the correlation of fragrant components and color patterns is less clear. We simultaneously used one way to address how floral color and fragrant formation in different tissues are generated during the development of an individual plant by transcriptome-based weighted gene co-expression network analysis (WGCNA). A spatiotemporal pattern variation of flavonols/carotenoids/chlorophyll pigmentation and benzenoid/phenylpropanoid/ monoterpene fragrant components between the tepal and corona in the flower tissues of Narcissus tazetta, was exhibited. Several candidate transcription factors: MYB12, MYB1, AP2-ERF, bZIP, NAC, MYB, C2C2, C2H2 and GRAS are shown to be associated with metabolite flux, the phenylpropanoid pathway to the production of flavonols/anthocyanin, as well as related to one branch of the phenylpropanoid pathway to the benzenoid/phenylpropanoid component in the tepal and the metabolite flux between the monoterpene and carotenoids biosynthesis pathway in coronas. It indicates that potential competition exists between floral pigment and floral fragrance during Narcissus tazetta individual plant development and evolutionary development.

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.

Attenuation of epigenetic regulator SMARCA4 and ERK-ETS signaling suppresses aging-related dopaminergic degeneration.

How complex interactions of genetic, environmental factors and aging jointly contribute to dopaminergic degeneration in Parkinson's disease (PD) is largely unclear. Here, we applied frequent gene co-expression analysis on human patient substantia nigra-specific microarray datasets to identify potential novel disease-related genes. In vivo Drosophila studies validated two of 32 candidate genes, a chromatin-remodeling factor SMARCA4 and a biliverdin reductase BLVRA. Inhibition of SMARCA4 was able to prevent aging-dependent dopaminergic degeneration not only caused by overexpression of BLVRA but also in four most common Drosophila PD models. Furthermore, down-regulation of SMARCA4 specifically in the dopaminergic neurons prevented shortening of life span caused by α-synuclein and LRRK2. Mechanistically, aberrant SMARCA4 and BLVRA converged on elevated ERK-ETS activity, attenuation of which by either genetic or pharmacological manipulation effectively suppressed dopaminergic degeneration in Drosophila in vivo. Down-regulation of SMARCA4 or drug inhibition of MEK/ERK also mitigated mitochondrial defects in PINK1 (a PD-associated gene)-deficient human cells. Our findings underscore the important role of epigenetic regulators and implicate a common signaling axis for therapeutic intervention in normal aging and a broad range of age-related disorders including PD.

H2O2 as a Feedback Signal on Dual-Located WHIRLY1 Associates with Leaf Senescence in Arabidopsis.

Leaf senescence, either as a natural stage of development or as an induced process under stress conditions, incorporates multiple intricate signaling pathways. At the cellular level, retrograde signals have been considered as important players during the initiation and progression of senescence in both animals and plants. The plant-specific single-strand DNA-binding protein WHIRLY1 (WHY1), a repressor of leaf natural senescence, is dually located in both nucleus and plastids. Despite many years of studies, the myth about its dual location and the underlying functional implications remain elusive. Here, we provide further evidence in Arabidopsis showing that alteration in WHY1 allocation between the nucleus and chloroplast causes perturbation in H2O2 homeostasis, resulting in adverse plant senescence phenotypes. The knockout of WHY1 increased H2O2 content at 37 days post-germination, coincident with an early leaf senescence phenotype, which can be rescued by ectopic expression of the nuclear isoform (nWHY1), but not by the plastid isoform (pWHY1). Instead, accumulated pWHY1 greatly provoked H2O2 in cells. On the other hand, exogenous H2O2 treatment induced a substantial plastid accumulation of WHY1 proteins and at the same time reduced the nuclear isoforms. This H2O2-induced loss of nucleus WHY1 isoform was accompanied by enhanced enrichments of histone H3 lysine 9 acetylation (H3K9ac) and recruitment of RNA polymerase II (RNAP II) globally, and specifically at the promoter of the senescence-related transcription factor WRKY53, which in turn activated WRKY53 transcription and led to a senescence phenotype. Thus, the distribution of WHY1 organelle isoforms and the feedback of H2O2 intervene in a circularly integrated regulatory network during plant senescence in Arabidopsis.

Genome-Wide Characterization and Gene Expression Analyses of GATA Transcription Factors in Moso Bamboo (Phyllostachys edulis).

Moso bamboo is well-known for its rapid-growth shoots and widespread rhizomes. However, the regulatory genes of these two processes are largely unexplored. GATA transcription factors regulate many developmental processes, but their roles in moso bamboo height control and rhizome development remains unexplored. Here, thirty-one bamboo GATA factors (PeGATAs) were identified, which are evolutionarily closer to rice than Arabidopsis, and their gene expression patterns were analyzed in bamboo development and phytohormone response with bioinformatics and molecular methods. Interestingly, PeGATAs could only be classified into three groups. Phytohormone responsive cis-elements were found in PeGATA promoters and the expression profiles showed that PeGATA genes might respond to gibberellin acid and abscisic acid but not to auxin at the transcriptional level. Furthermore, PeGATA genes have a tissue-specific expression pattern in bamboo rhizomes. Interestingly, most PeGATA genes were down-regulated during the rapid-growth of bamboo shoots. In addition, over-expressing one of the PeGATA genes, PeGATA26, significantly repressed the primary root length and plant height of transgenic Arabidopsis plants, which may be achieved by promoting the gibberellin acid turnover. Overall, our results provide insight into the function of GATA transcription factors in bamboo, and into genetic resources for engineering plant height.

WHIRLY1 Occupancy Affects Histone Lysine Modification and WRKY53 Transcription in Arabidopsis Developmental Manner.

Single-stranded DNA-binding proteins (SSBs) are assumed to involve in DNA replication, DNA repairmen, and gene transcription. Here, we provide the direct evidence on the functionality of an Arabidopsis SSB, WHIRLY1, by using loss- or gain-of-function lines. We show that WHIRLY1 binding to the promoter of WRKY53 represses the enrichment of H3K4me3, but enhances the enrichment of H3K9ac at the region contained WHIRLY1-binding sequences and TATA box or the translation start region of WRKY53, coincided with a recruitment of RNAPII. In vitro ChIP assays confirm that WHIRLY1 inhibits H3K4me3 enrichment at the preinitiation complex formation stage, while promotes H3K9ac enrichment and RNAPII recruitment at the elongation stage, consequently affecting the transcription of WRKY53. These results further explore the molecular actions underlying SSB-mediated gene transcription through epigenetic regulation in plant senescence.

Isolation, Purification, and Detection of Micro RNAs in Plant Senescence.

Micro RNAs (miRNAs) are small noncoding RNA molecules that function in transcriptional level to regulate gene expression both in plants and animals. Increasing researches have shown that miRNAs are key regulators in plant development and stress responses, and emerging evidence indicates the potential role of miRNAs on plant senescence. In this chapter we summarize the daily methods used for identification and study of miRNAs in plants, including the isolation of total RNA, the purification of miRNAs, and the methods used to detect miRNAs in plants. The committed steps or modifications of these methods used in plant senescence research are noted.

Dual-Located WHIRLY1 Interacting with LHCA1 Alters Photochemical Activities of Photosystem I and Is Involved in Light Adaptation in Arabidopsis.

Plastid-nucleus-located WHIRLY1 protein plays a role in regulating leaf senescence and is believed to associate with the increase of reactive oxygen species delivered from redox state of the photosynthetic electron transport chain. In order to make sure whether WHIRLY1 plays a role in photosynthesis, in this study, the performances of photosynthesis were detected in Arabidopsis whirly1 knockout (kowhy1) and plastid localized WHIRLY1 overexpression (oepWHY1) plants. Loss of WHIRLY1 leads to a higher photochemical quantum yield of photosystem I Y(I) and electron transport rate (ETR) and a lower non-photochemical quenching (NPQ) involved in the thermal dissipation of excitation energy of chlorophyll fluorescence than the wild type. Further analyses showed that WHIRLY1 interacts with Light-harvesting protein complex I (LHCA1) and affects the expression of genes encoding photosystem I (PSI) and light harvest complexes (LHCI). Moreover, loss of WHIRLY1 decreases chloroplast NAD(P)H dehydrogenase-like complex (NDH) activity and the accumulation of NDH supercomplex. Several genes encoding the PSI-NDH complexes are also up-regulated in kowhy1 and the whirly1whirly3 double mutant (ko1/3) but steady in oepWHY1 plants. However, under high light conditions (800 µmol m-2 s-1), both kowhy1 and ko1/3 plants show lower ETR than wild-type which are contrary to that under normal light condition. Moreover, the expression of several PSI-NDH encoding genes and ERF109 which is related to jasmonate (JA) response varied in kowhy1 under different light conditions. These results indicate that WHIRLY1 is involved in the alteration of ETR by affecting the activities of PSI and supercomplex formation of PSI with LHCI or NDH and may acting as a communicator between the plastids and the nucleus.

Structure of Pigment Metabolic Pathways and Their Contributions to White Tepal Color Formation of Chinese Narcissus tazetta var. chinensis cv Jinzhanyintai.

Chinese narcissus (Narcissus tazetta var. chinensis) is one of the ten traditional flowers in China and a famous bulb flower in the world flower market. However, only white color tepals are formed in mature flowers of the cultivated varieties, which constrains their applicable occasions. Unfortunately, for lack of genome information of narcissus species, the explanation of tepal color formation of Chinese narcissus is still not clear. Concerning no genome information, the application of transcriptome profile to dissect biological phenomena in plants was reported to be effective. As known, pigments are metabolites of related metabolic pathways, which dominantly decide flower color. In this study, transcriptome profile and pigment metabolite analysis methods were used in the most widely cultivated Chinese narcissus "Jinzhanyintai" to discover the structure of pigment metabolic pathways and their contributions to white tepal color formation during flower development and pigmentation processes. By using comparative KEGG pathway enrichment analysis, three pathways related to flavonoid, carotenoid and chlorophyll pigment metabolism showed significant variations. The structure of flavonoids metabolic pathway was depicted, but, due to the lack of F3'5'H gene; the decreased expression of C4H, CHS and ANS genes; and the high expression of FLS gene, the effect of this pathway to synthesize functional anthocyanins in tepals was weak. Similarly, the expression of DXS, MCT and PSY genes in carotenoids synthesis sub-pathway was decreased, while CCD1/CCD4 genes in carotenoids degradation sub-pathway was increased; therefore, the effect of carotenoids metabolic pathway to synthesize adequate color pigments in tepals is restricted. Interestingly, genes in chlorophyll synthesis sub-pathway displayed uniform down-regulated expression, while genes in heme formation and chlorophyll breakdown sub-pathways displayed up-regulated expression, which also indicates negative regulation of chlorophyll formation. Further, content change trends of various color metabolites detected by HPLC in tepals are consistent with the additive gene expression patterns in each pathway. Therefore, all three pathways exhibit negative control of color pigments synthesis in tepals, finally resulting in the formation of white tepals. Interestingly, the content of chlorophyll was more than 10-fold higher than flavonoids and carotenoids metabolites, which indicates that chlorophyll metabolic pathway may play the major role in deciding tepal color formation of Chinese narcissus.

Phosphorylation of WHIRLY1 by CIPK14 Shifts Its Localization and Dual Functions in Arabidopsis.

Plastid-to-nucleus retrograde signaling is critical for normal growth and development in plants. The dual-function and dual-located ssDNA binding protein WHIRLY1 (WHY1) has been proposed to coordinate the retrograde signaling from plastids to the nucleus. However, the regulatory mechanism governing the functional switch of WHY1 for mediating plastid-to-nucleus retrograde signaling remains unknown. Here, we report that the Calcineurin B-Like-Interacting Protein Kinase14 (CIPK14) interacts with and phosphorylates WHY1 in Arabidopsis. Phosphorylation of WHY1 results in increased accumulation in the nucleus and enhanced binding with the promoter of WRKY53, which encodes a key transcription factor regulating leaf senescence in Arabidopsis. Transgenic plants overexpressing CIPK14 showed an increased nuclear isoform but decreased plastid isoform of WHY1, among which 95% of transgenic lines showed the stay-green phenotype and 5% of lines showed the variegated pale-green phenotype. Interestingly, the phenotypes of both types of transgenic plants could be recovered by overexpression of plastid-form WHY1. In contrast, knockdown of CIPK14 caused early senescence and even seedling-lethal phenotypes along with elevated expression of senescence-related genes such as WRKY53, SAG12, and NDHF but decreased expression of MER11, RAD50, and POR genes, which could be rescued by overexpression of CIPK14 but not by overexpressing plastid-form or nuclear-form WHY1; the stay-green plants overexpressing CIPK14 showed reduced expression of WRKY53, SAG12, NDHF, and large plastid rRNA. Consistently, the accumulation of nuclear-form WHY1 was significantly reduced in the CIPK14 knockdown lines, resulting in a low ratio of nuclear-/plastid-form WHY1. Taken together, our results demonstrate that CIPK14 regulates the phosphorylation and organellar distributions of WHY1 and pinpoint that CIPK14 may function as a cellular switch between leaf senescence and plastid development for coordinating the intercellular signaling in Arabidopsis.

The single-stranded DNA-binding protein WHIRLY1 represses WRKY53 expression and delays leaf senescence in a developmental stage-dependent manner in Arabidopsis.

Leaf senescence in plants involves both positive and negative transcriptional regulation. In this work, we show evidence for the single-stranded DNA-binding protein WHIRLY1 (WHY1) that functions as an upstream suppressor of WRKY53 in a developmental stage-dependent manner during leaf senescence in Arabidopsis (Arabidopsis thaliana). The why1 mutant displayed an early-senescence phenotype. In this background, the expression levels of both WRKY53 and the senescence-associated protease gene SAG12 increased. WHY1 bound to the sequence region that contains an elicitor response element motif-like sequence, GNNNAAATT, plus an AT-rich telomeric repeat-like sequence in the WRKY53 promoter in in vivo and in vitro mutagenesis assays as well as in a chromatin immunoprecipitation assay. This binding to the promoter of WRKY53 was regulated in a developmental stage-dependent manner, as verified by chromatin immunoprecipitation-polymerase chain reaction assay. This direct interaction was further determined by a transient expression assay in which WHY1 repressed ß-GLUCURONIDASE gene expression driven by the WRKY53 promoter. Genetic analysis of double mutant transgenic plants revealed that WHY1 overexpression in the wrky53 mutant (oeWHY1wrky53) had no effect on the stay-green phenotype of the wrky53 mutant, while a WHY1 knockout mutant in the wrky53 mutant background (why1wrky53) generated subtle change in the leaf yellow/green phenotype. These results suggest that WHY1 was an upstream regulator of WRKY53 during leaf senescence.

Type 4 metallothionein genes are involved in regulating Zn ion accumulation in late embryo and in controlling early seedling growth in Arabidopsis.

Type 4 metallothionein (MT) genes are recognized for their specific expression in higher plant seeds, but their functions are still unclear. In this study, the functions of two Arabidopsis metallothionein genes, AtMT4a and AtMT4b, are investigated in seed development, germination and early seedling growth. Transcriptional analysis showed that these two genes are specifically expressed in late embryos. Subcellular localization displayed that both AtMT4a and AtMT4b are widespread distributed in cytoplasm, nucleus and membrane. Co-silencing RNAi of AtMT4a and AtMT4b reduced seed weight and influenced the early seedling growth after germination, whereas overexpression of these two genes caused the opposite results. Detailed analysis showed clearly the correlation of AtMT4a and AtMT4b to the accumulation of some important metal ions in late embryos, especially to Zn ion storing in seeds, which then serves as part of early Zn ion resources for post-germinated seedling growth. Furthermore, phytohormone abscisic acid (ABA) and gibberellic acid (GA) may play roles in regulating the expression and function of AtMT4a and AtMT4b during seed development; and this may influence Zn accumulation in seeds and Zn ion nutrient supplementation in the early seedling growth after germination.