The ABA-AtNAP-SAG113 PP2C module regulates leaf senescence by dephoshorylating SAG114 SnRK3.25 in Arabidopsis.

We previously reported that ABA inhibits stomatal closure through AtNAP-SAG113 PP2C regulatory module during leaf senescence. The mechanism by which this module exerts its function is unknown. Here we report the identification and functional analysis of SAG114, a direct target of the regulatory module. SAG114 encodes SnRK3.25. Both bimolecular fluorescence complementation (BiFC) and yeast two-hybrid assays show that SAG113 PP2C physically interacts with SAG114 SnRK3.25. Biochemically the SAG113 PP2C dephosphorylates SAG114 in vitro and in planta. RT-PCR and GUS reporter analyses show that SAG114 is specifically expressed in senescing leaves in Arabidopsis. Functionally, the SAG114 knockout mutant plants have a significantly bigger stomatal aperture and a much faster water loss rate in senescing leaves than those of wild type, and display a precocious senescence phenotype. The premature senescence phenotype of sag114 is epistatic to sag113 (that exhibits a remarkable delay in leaf senescence) because the sag113 sag114 double mutant plants show an early leaf senescence phenotype, similar to that of sag114. These results not only demonstrate that the ABA-AtNAP-SAG113 PP2C regulatory module controls leaf longevity by dephosphorylating SAG114 kinase, but also reveal the involvement of the SnRK3 family gene in stomatal movement and water loss during leaf senescence.

Ethylene controls cambium stem cell activity via promoting local auxin biosynthesis.

The vascular cambium is the main secondary meristem in plants that produces secondary phloem (outside) and xylem (inside) on opposing sides of the cambium. The phytohormone ethylene has been implicated in vascular cambium activity, but the regulatory network underlying ethylene-mediated cambial activity remains to be elucidated. Here, we found that PETAL MOVEMENT-RELATED PROTEIN1 (RhPMP1), an ethylene-inducible HOMEODOMAIN-LEUCINE ZIPPER I transcription factor in woody plant rose (Rosa hybrida), regulates local auxin biosynthesis and auxin transport to maintain cambial activity. Knockdown of RhPMP1 resulted in smaller midveins and reduced auxin content, while RhPMP1 overexpression resulted in larger midveins and increased auxin levels compared with the wild-type plants. Furthermore, we revealed that Indole-3-pyruvate monooxygenase YUCCA 10 (RhYUC10) and Auxin transporter-like protein 2 (RhAUX2), encoding an auxin biosynthetic enzyme and an auxin influx carrier, respectively, are direct downstream targets of RhPMP1. In summary, our results suggest that ethylene promotes an auxin maximum in the cambium adjacent to the xylem to maintain cambial activity.

Warming-induced changes of broccoli head to cauliflower-like curd in Brassica oleracea are regulated by DNA methylation as revealed by methylome and transcriptome co-profiling.

Increasingly warming temperature impacts on all aspects of growth and development in plants. Flower development is a complex process that is very sensitive to ambient temperature, and warming temperatures often lead to abnormal flower development and remarkably reduce the quality and yield of inflorescent vegetables and many other crops, which can be exemplified by Brassica oleracea cv. Green Harmony F1, a broccoli cultivar, whose floral development is ceased at inflorescence meristem (at 28 °C) or floral primordium stage (at 22 °C), forming a cauliflower-like curd (28 °C) or intermediate curd (22 °C) instead of normal broccoli head at 16 °C. However, the underlying molecular regulatory mechanisms are not well understood. Here we report that warming temperature (28 °C or 22 °C) induced hypermethylation of the genome, especially the promoter regions of such sets of genes as ribosome biogenesis-related and others, leading to the suppression of the apex-highly-expressed distinctive genes, subsequently resulting in the abnormal floral development, as revealed by methylome and transcriptome co-profiling. The regulation of warming-induced abnormal floral development in broccoli was further verified by the fact that the DNA methylation inhibitor 5-azacytidine (5-azaC) released the expression of genes from the warming temperature-induced suppression, and restored the broccoli development to normalcy at warming temperature. The research provided new approaches to breeding broccoli and other crops for growing in wider or warmer temperature zones. Graphical Abstract.

A positive feedback regulatory loop, SA-AtNAP-SAG202/SARD1-ICS1-SA, in SA biosynthesis involved in leaf senescence but not defense response.

Salicylic acid (SA) is an important plant hormone that regulates defense responses and leaf senescence. It is imperative to understand upstream factors that regulate genes of SA biosynthesis. SAG202/SARD1 is a key regulator for isochorismate synthase 1 (ICS1) induction and SA biosynthesis in defense responses. The regulatory mechanism of SA biosynthesis during leaf senescence is not well understood. Here we show that AtNAP, a senescence-specific NAC family transcription factor, directly regulates a senescence-associated gene named SAG202 as revealed in yeast one-hybrid and in planta assays. Inducible overexpreesion of AtNAP and SAG202 lead to high levels of SA and precocious senescence in leaves. Individual knockout mutants of sag202 and ics1 have markedly reduced SA levels and display a significantly delayed leaf senescence phenotype. Furthermore, SA positively feedback regulates AtNAP and SAG202. Our research has uncovered a unique positive feedback regulatory loop, SA-AtNAP-SAG202-ICS1-SA, that operates to control SA biosynthesis associated with leaf senescence but not defense response.

The circadian-controlled PIF8-BBX28 module regulates petal senescence in rose flowers by governing mitochondrial ROS homeostasis at night.

Reactive oxygen species (ROS) are unstable reactive molecules that are toxic to cells. Regulation of ROS homeostasis is crucial to protect cells from dysfunction, senescence, and death. In plant leaves, ROS are mainly generated from chloroplasts and are tightly temporally restricted by the circadian clock. However, little is known about how ROS homeostasis is regulated in nonphotosynthetic organs, such as petals. Here, we showed that hydrogen peroxide (H2O2) levels exhibit typical circadian rhythmicity in rose (Rosa hybrida) petals, consistent with the measured respiratory rate. RNA-seq and functional screening identified a B-box gene, RhBBX28, whose expression was associated with H2O2 rhythms. Silencing RhBBX28 accelerated flower senescence and promoted H2O2 accumulation at night in petals, while overexpression of RhBBX28 had the opposite effects. RhBBX28 influenced the expression of various genes related to respiratory metabolism, including the TCA cycle and glycolysis, and directly repressed the expression of SUCCINATE DEHYDROGENASE 1, which plays a central role in mitochondrial ROS (mtROS) homeostasis. We also found that PHYTOCHROME-INTERACTING FACTOR8 (RhPIF8) could activate RhBBX28 expression to control H2O2 levels in petals and thus flower senescence. Our results indicate that the circadian-controlled RhPIF8-RhBBX28 module is a critical player that controls flower senescence by governing mtROS homeostasis in rose.

Ethylene-regulated asymmetric growth of the petal base promotes flower opening in rose (Rosa hybrida).

Flowers are the core reproductive structures and key distinguishing features of angiosperms. Flower opening to expose stamens and gynoecia is important in cases where pollinators much be attracted to promote cross-pollination, which can enhance reproductive success and species preservation. The floral opening process is accompanied by the coordinated movement of various floral organs, particularly petals. However, the mechanisms underlying petal movement and flower opening are not well understood. Here, we integrated anatomical, physiological, and molecular approaches to determine the petal movement regulatory network using rose (Rosa hybrida) as a model. We found that PETAL MOVEMENT-RELATED PROTEIN1 (RhPMP1), a homeodomain transcription factor (TF) gene, is a direct target of ETHYLENE INSENSITIVE3, a TF that functions downstream of ethylene signaling. RhPMP1 expression was upregulated by ethylene and specifically activated endoreduplication of parenchyma cells on the adaxial side of the petal (ADSP) base by inducing the expression of RhAPC3b, a gene encoding the core subunit of the Anaphase-Promoting Complex. Cell expansion of the parenchyma on the ADSP base was subsequently enhanced, thus resulting in asymmetric growth of the petal base, leading to the typical epinastic movement of petals and flower opening. These findings provide insights into the pathway regulating petal movement and associated flower-opening mechanisms.�.

AUXIN RESPONSE FACTOR 18-HISTONE DEACETYLASE 6 module regulates floral organ identity in rose (Rosa hybrida).

The phytohormone auxin plays a pivotal role in floral meristem initiation and gynoecium development, but whether and how auxin controls floral organ identity remain largely unknown. Here, we found that auxin levels influence organ specification, and changes in auxin levels influence homeotic transformation between petals and stamens in rose (Rosa hybrida). The PIN-FORMED-LIKES (PILS) gene RhPILS1 governs auxin levels in floral buds during floral organogenesis. RhAUXIN RESPONSE FACTOR 18 (RhARF18), whose expression decreases with increasing auxin content, encodes a transcriptional repressor of the C-class gene RhAGAMOUS (RhAG), and controls stamen-petal organ specification in an auxin-dependent manner. Moreover, RhARF18 physically interacts with the histone deacetylase (HDA) RhHDA6. Silencing of RhHDA6 increases H3K9/K14 acetylation levels at the site adjacent to the RhARF18-binding site in the RhAG promoter and reduces petal number, indicating that RhARF18 might recruit RhHDA6 to the RhAG promoter to reinforce the repression of RhAG transcription. We propose a model for how auxin homeostasis controls floral organ identity via regulating transcription of RhAG.

The leaf senescence-promoting transcription factor AtNAP activates its direct target gene CYTOKININ OXIDASE 3 to facilitate senescence processes by degrading cytokinins.

Cytokinins (CKs) are a class of adenine-derived plant hormones that plays pervasive roles in plant growth and development including cell division, morphogenesis, lateral bud outgrowth, leaf expansion and senescence. CKs as a "fountain of youth" prolongs leaf longevity by inhibiting leaf senescence, and therefore must be catabolized for senescence to occur. AtNAP, a senescence-specific transcription factor has a key role in promoting leaf senescence. The role of AtNAP in regulating CK catabolism is unknown. Here we report the identification and characterization of AtNAP-AtCKX3 (cytokinin oxidase 3) module by which CKs are catabolized during leaf senescence in Arabidopsis. Like AtNAP, AtCKX3 is highly upregulated during leaf senescence. When AtNAP is chemically induced AtCKX3 is co-induced; and when AtNAP is knocked out, the expression of AtCKX3 is abolished. AtNAP physically binds to the cis element of the AtCKX3 promoter to direct its expression as revealed by yeast one-hybrid assays and in planta experiments. Leaves of the atckx3 knockout lines have higher CK concentrations and a delayed senescence phenotype compared with those of WT. In contrast, leaves with inducible expression of AtCKX3 have lower CK concentrations and exhibit a precocious senescence phenotype compared with WT. This research reveals that AtNAP transcription factor-AtCKX3 module regulates leaf senescence by connecting two antagonist plant hormones abscisic acid and CKs.

Regulation of Leaf Longevity by DML3-Mediated DNA Demethylation.

Leaf senescence is driven by the expression of senescence-associated genes (SAGs). Development-specific genes often undergo DNA demethylation in their promoter and other regions, which regulates gene expression. Whether and how DNA demethylation regulates the expression of SAGs and thus leaf senescence remain elusive. Whole-genome bisulfite sequencing (WGBS) analyses of wild-type (WT) and demeter-like 3 (dml3) Arabidopsis leaves at three developmental stages revealed hypermethylation during leaf senescence in dml3 compared with WT, and 20 556 differentially methylated regions (DMRs) were identified by comparing the methylomes of dml3 and WT in the CG, CHG, and CHH contexts. Furthermore, we identified that 335 DMR-associated genes (DMGs), such as NAC016 and SEN1, are upregulated during leaf senescence, and found an inverse correlation between the DNA methylation levels (especially in the promoter regions) and the transcript abundances of the related SAGs in WT. In contrast, in dml3 the promoters of SAGs were hypermethylated and their transcript levels were remarkably reduced, and leaf senescence was significantly delayed. Collectively, our study unraveled a novel epigenetic regulatory mechanism underlying leaf senescence in which DML3 is expressed at the onset of and during senescence to demethylate promoter, gene body or 3' UTR regions to activate a set of SAGs.

In rose, transcription factor PTM balances growth and drought survival via PIP2;1 aquaporin.

Plants have evolved sophisticated systems in response to environmental changes, and growth arrest is a common strategy used to enhance stress tolerance. Despite the growth-survival trade-off being essential to the shaping of plant productivity, the mechanisms balancing growth and survival remain largely unknown. Aquaporins play a crucial role in growth and stress responses by controlling water transport across membranes. Here, we show that RhPIP2;1, an aquaporin from rose (Rosa sp.), interacts with a membrane-tethered MYB protein, RhPTM. Water deficiency triggers nuclear translocation of the RhPTM C terminus. Silencing of RhPTM causes continuous growth under drought stress and a consequent decrease in survival rate. RNA sequencing (RNA-seq) indicated that RhPTM influences the expression of genes related to carbohydrate metabolism. Water deficiency induces phosphorylation of RhPIP2;1 at Ser 273, which is sufficient to promote nuclear translocation of the RhPTM C terminus. These results indicate that the RhPIP2;1-RhPTM module serves as a key player in orchestrating the trade-off between growth and stress survival in Rosa.

Maintenance of grafting-induced epigenetic variations in the asexual progeny of Brassica oleracea and B. juncea chimera.

Grafting-induced variations have been observed in many plant species, but the heritability of variation in progeny is not well understood. In our study, adventitious shoots from the C cell lineage of shoot apical meristem (SAM) grafting chimera TCC (where the origin of the outmost, middle and innermost cell layers, respectively, of SAM is designated by 'T' for tuber mustard and 'C' for red cabbage) were induced and identified as r-CCC (r = regenerated). To investigate the maintenance of grafting variations during cell propagation and regeneration, different generations of asexual progeny (r-CCCn, n = generation) were established through successive regeneration of axillary shoots from r-CCC. The fourth generation of r-CCC (r-CCC4) was selected to perform whole genome bisulfite sequencing for comparative analysis of hetero-grafting-induced global methylation changes relative to r-s-CCC4 (s = self-grafting). Increased CHH methylation levels and proportions were observed in r-CCC4, with substantial changes occurring in the repeat elements. Small RNA sequencing revealed 1135 specific small interfering RNA (siRNA) tags that were typically expressed in r-CCC, r-CCC2 and r-CCC4. Notably, 65% of these specific siRNAs were associated with repeat elements, termed RE siRNAs. Subsequent analysis revealed that the CHH methylation of RE siRNA-overlapping regions was mainly hypermethylation in r-CCC4, indicating that they were responsible for directing and maintaining grafting-induced CHH methylation. Moreover, the expression of 13 differentially methylated genes (DMGs) correlated with the phenotypic variation, showing differential expression levels between r-CCC4 and r-s-CCC4. These DMGs were predominantly CG hypermethylated, their methylation modifications corresponded to the transcription of relative methyltransferase.

Concepts and Types of Senescence in Plants.

Concepts, classification, and the relationship between different types of senescence are discussed in this chapter. Senescence-related terminology frequently used in yeast, animal, and plant systems and senescence processes at cellular, organ, and organismal levels are clarified.

S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis.

The phytohormone salicylic acid (SA) plays essential roles in biotic and abiotic responses, plant development, and leaf senescence. 2,5-Dihydroxybenzoic acid (2,5-DHBA or gentisic acid) is one of the most commonly occurring aromatic acids in green plants and is assumed to be generated from SA, but the enzymes involved in its production remain obscure. DMR6 (Downy Mildew Resistant6; At5g24530) has been proven essential in plant immunity of Arabidopsis (Arabidopsis thaliana), but its biochemical properties are not well understood. Here, we report the discovery and functional characterization of DMR6 as a salicylic acid 5-hydroxylase (S5H) that catalyzes the formation of 2,5-DHBA by hydroxylating SA at the C5 position of its phenyl ring in Arabidopsis. S5H/DMR6 specifically converts SA to 2,5-DHBA in vitro and displays higher catalytic efficiency (Kcat/Km = 4.96 × 104 m-1 s-1) than the previously reported S3H (Kcat/Km = 6.09 × 103 m-1 s-1) for SA. Interestingly, S5H/DMR6 displays a substrate inhibition property that may enable automatic control of its enzyme activities. The s5h mutant and s5hs3h double mutant overaccumulate SA and display phenotypes such as a smaller growth size, early senescence, and a loss of susceptibility to Pseudomonas syringae pv tomato DC3000. S5H/DMR6 is sensitively induced by SA/pathogen treatment and is expressed widely from young seedlings to senescing plants, whereas S3H is more specifically expressed at the mature and senescing stages. Collectively, our results disclose the identity of the enzyme required for 2,5-DHBA formation and reveal a mechanism by which plants fine-tune SA homeostasis by mediating SA 5-hydroxylation.

Differential DNA methylation and gene expression in reciprocal hybrids between Solanum lycopersicum and S. pimpinellifolium.

Wide hybridization is a common and efficient breeding strategy for enhancing crop yield and quality. An interesting phenomenon is that the reciprocal hybrids usually show different phenotypes, and its underlying mechanism is not well understood. Here, we reported our comparative analysis of the DNA methylation patterns in Solanum lycopersicum, Solanum pimpinellifolium and their reciprocal hybrids by methylated DNA immunoprecipitation sequencing. The reciprocal hybrids had lower levels of DNA methylation in CpG islands and LTR retroelements when compared with those of their parents. Importantly, remarkable differences in DNA methylation patterns, mainly in introns and CDS regions, were revealed between the reciprocal hybrids. These different methylated regions were mapped to 79 genes, 14 of which were selected for analysis of gene expression levels. While there was an inverse correlation between DNA methylation and gene expression in promoter regions, the relationship was complicated in gene body regions. Further association analysis revealed that there were 15 differentially methylated genes associated with siRNAs, and that the methylation levels of these genes were inversely correlated with respective siRNAs. All these data raised the possibility that the direction of hybridization induced the divergent epigenomes leading to changes in the transcription levels of reciprocal hybrids.

Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes.

Red dragon fruit or red pitaya (Hylocereus polyrhizus) is the only edible fruit that contains betalains. The color of betalains ranges from red and violet to yellow in plants. Betalains may also serve as an important component of health-promoting and disease-preventing functional food. Currently, the biosynthetic and regulatory pathways for betalain production remain to be fully deciphered. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses were used to reveal the molecular mechanism of betalain biosynthesis in H. polyrhizus fruits at white and red pulp stages, respectively. A total of 1946 proteins were identified as the differentially expressed between the two samples, and 936 of them were significantly highly expressed at the red pulp stage of H. polyrhizus. RNA-seq and iTRAQ analyses showed that some transcripts and proteins were positively correlated; they belonged to "phenylpropanoid biosynthesis", "tyrosine metabolism", "flavonoid biosynthesis", "ascorbate and aldarate metabolism", "betalains biosynthesis" and "anthocyanin biosynthesis". In betalains biosynthesis pathway, several proteins/enzymes such as polyphenol oxidase, CYP76AD3 and 4,5-dihydroxy-phenylalanine (DOPA) dioxygenase extradiol-like protein were identified. The present study provides a new insight into the molecular mechanism of the betalain biosynthesis at the posttranscriptional level.

A novel NAP member GhNAP is involved in leaf senescence in Gossypium hirsutum.

Premature leaf senescence has a negative influence on the yield and quality of cotton, and several genes have been found to regulate leaf senescence. Howeer, many underlying transcription factors are yet to be identified. In this study, a NAP-like transcription factor (GhNAP) was isolated from Gossypium hirsutum. GhNAP has the typical NAC structure and a conserved novel subdomain in its divergent transcription activation region (TAR). GhNAP was demonstrated to be a nuclear protein, and it showed transcriptional activation activity in yeast. Furthermore, the expression of GhNAP was closely associated with leaf senescence. GhNAP could rescue the delayed-senescence phenotype of the atnap null mutant. Overexpression of GhNAP could cause precocious senescence in Arabidopsis. However, down-regulation of GhNAP delayed leaf senescence in cotton, and affected cotton yield and its fibre quality. Moreover, the expression of GhNAP can be induced by abscisic acid (ABA), and the delayed leaf senescence phenotype in GhNAPi plants might be caused by the decreased ABA level and reduced expression level of ABA-responsive genes. All of the results suggested that GhNAP could regulate the leaf senescence via the ABA-mediated pathways and was further related to the yield and quality in cotton.