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.�.

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.

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.

Salicylic acid 3-hydroxylase regulates Arabidopsis leaf longevity by mediating salicylic acid catabolism.

The plant hormone salicylic acid (SA) plays critical roles in plant defense, stress responses, and senescence. Although SA biosynthesis is well understood, the pathways by which SA is catabolized remain elusive. Here we report the identification and characterization of an SA 3-hydroxylase (S3H) involved in SA catabolism during leaf senescence. S3H is associated with senescence and is inducible by SA and is thus a key part of a negative feedback regulation system of SA levels during senescence. The enzyme converts SA (with a Km of 58.29 µM) to both 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-DHBA in vitro but only 2,3-DHBA in vivo. The s3h knockout mutants fail to produce 2,3-DHBA sugar conjugates, accumulate very high levels of SA and its sugar conjugates, and exhibit a precocious senescence phenotype. Conversely, the gain-of-function lines contain high levels of 2,3-DHBA sugar conjugates and extremely low levels of SA and its sugar conjugates and display a significantly extended leaf longevity. This research reveals an elegant SA catabolic mechanism by which plants regulate SA levels by converting it to 2,3-DHBA to prevent SA overaccumulation. The research also provides strong molecular genetic evidence for an important role of SA in regulating the onset and rate of leaf senescence.

SAUR36, a small auxin up RNA gene, is involved in the promotion of leaf senescence in Arabidopsis.

Small Auxin Up RNA genes (SAURs) are early auxin-responsive genes, but whether any of them are involved in leaf senescence is not known. Auxin, on the other hand, has been shown to have a role in leaf senescence. Some of the external application experiments indicated that auxin can inhibit leaf senescence, whereas other experiments indicated that auxin can promote leaf senescence. Here, we report the identification and characterization of an Arabidopsis (Arabidopsis thaliana) leaf senescence-associated gene named SAG201, which is highly up-regulated during leaf senescence and can be induced by 1-naphthaleneacetic acid, a synthetic auxin. It encodes a functionally uncharacterized SAUR that has been annotated as SAUR36. Leaf senescence in transfer DNA insertion saur36 knockout lines was delayed as revealed by analyses of chlorophyll content, F(v)/F(m) ratio (a parameter for photosystem II activity), ion leakage, and the expression of leaf senescence marker genes. In contrast, transgenic Arabidopsis plants overexpressing SAUR36 (without its 3' untranslated region [UTR]) displayed an early leaf senescence phenotype. However, plants overexpressing SAUR36 with its 3' UTR were normal and did not exhibit the early-senescence phenotype. These data suggest that SAUR36 is a positive regulator of leaf senescence and may mediate auxin-induced leaf senescence and that the 3' UTR containing a highly conserved downstream destabilizes the SAUR36 transcripts in young leaves.

Translational researches on leaf senescence for enhancing plant productivity and quality.

Leaf senescence is a very important trait that limits yield and biomass accumulation of agronomic crops and reduces post-harvest performance and the nutritional value of horticultural crops. Significant advance in physiological and molecular understanding of leaf senescence has made it possible to devise ways of manipulating leaf senescence for agricultural improvement. There are three major strategies in this regard: (i) plant hormone biology-based leaf senescence manipulation technology, the senescence-specific gene promoter-directed IPT system in particular; (ii) leaf senescence-specific transcription factor biology-based technology; and (iii) translation initiation factor biology-based technology. Among the first strategy, the P SAG12 -IPT autoregulatory senescence inhibition system has been widely explored and successfully used in a variety of plant species for manipulating senescence. The vast majority of the related research articles (more than 2000) showed that crops harbouring the autoregulatory system displayed a significant delay in leaf senescence without any abnormalities in growth and development, a marked increase in grain yield and biomass, dramatic improvement in horticultural performance, and/or enhanced tolerance to drought stress. This technology is approaching commercialization. The transcription factor biology-based and translation initiation factor biology-based technologies have also been shown to be very promising and have great potentials for manipulating leaf senescence in crops. Finally, it is speculated that technologies based on the molecular understanding of nutrient recycling during leaf senescence are highly desirable and are expected to be developed in future translational leaf senescence research.

An ABA-regulated and Golgi-localized protein phosphatase controls water loss during leaf senescence in Arabidopsis.

It is known that a senescing leaf loses water faster than a non-senescing leaf and that ABA has an important role in promoting leaf senescence. However, questions such as why water loss is faster, how water loss is regulated, and how ABA functions in leaf senescence are not well understood. Here we report on the identification and functional analysis of a leaf senescence associated gene called SAG113. The RNA blot and GUS reporter analyses all show that SAG113 is expressed in senescing leaves and is induced by ABA in Arabidopsis. The SAG113 expression levels are significantly reduced in aba2 and abi4 mutants. A GFP fusion protein analysis revealed that SAG113 protein is localized in the Golgi apparatus. SAG113 encodes a protein phosphatase that belongs to the PP2C family and is able to functionally complement a yeast PP2C-deficient mutant TM126 (ptc1Δ). Leaf senescence is delayed in the SAG113 knockout mutant compared with that in the wild type, stomatal movement in the senescing leaves of SAG113 knockouts is more sensitive to ABA than that of the wild type, and the rate of water loss in senescing leaves of SAG113 knockouts is significantly reduced. In contrast, inducible over-expression of SAG113 results in a lower sensitivity of stomatal movement to ABA treatment, more rapid water loss, and precocious leaf senescence. No other aspects of growth and development, including seed germination, were observed. These findings suggest that SAG113, a negative regulator of ABA signal transduction, is specifically involved in the control of water loss during leaf senescence.

An abscisic acid-AtNAP transcription factor-SAG113 protein phosphatase 2C regulatory chain for controlling dehydration in senescing Arabidopsis leaves.

AtNAP is a NAC family transcription factor gene that plays a key role in leaf senescence but its underlying mechanisms are not known. SENESCENCE-ASSOCIATED GENE113 (SAG113), a gene encoding a Golgi-localized protein phosphatase 2C family protein phosphatase, mediates abscisic acid (ABA)-regulated stomatal movement and water loss specifically during leaf senescence. Here we report that SAG113 is a direct target gene of the AtNAP transcription factor. We found that both AtNAP and SAG113 were induced by leaf senescence and ABA. When AtNAP was chemically induced, SAG113 was also induced whereas when AtNAP was knocked out, the ABA- and senescence-induced expression of SAG113 was reduced. These data suggest that the expression of SAG113 is predominantly dependent on AtNAP. Functionally, overexpression of SAG113 restored the markedly delayed leaf senescence phenotype in atnap knockouts to wild type. Yeast (Saccharomyces cerevisiae) one-hybrid experiments and electrophoresis mobility shift assays showed that AtNAP could physically bind to the SAG113 promoter in vivo and in vitro, respectively. Site-directed mutagenesis revealed that AtNAP binds to a 9-bp core sequence of the SAG113 promoter, 5'CACGTAAGT3'. These results indicate that there is a unique regulatory chain, ABA-AtNAP-SAG113 protein phosphastase 2C, which controls stomatal movement and water loss during leaf senescence.

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.

Convergence and divergence in gene expression profiles induced by leaf senescence and 27 senescence-promoting hormonal, pathological and environmental stress treatments.

In addition to age and developmental progress, leaf senescence and senescence-associated genes (SAGs) can be induced by other factors such as plant hormones, pathogen infection and environmental stresses. The relationship is not clear, however, between these induced senescence processes and developmental leaf senescence, and to what extent these senescence-promoting signals mimic age and developmental senescence in terms of gene expression profiles. By analysing microarray expression data from 27 different treatments (that are known to promote senescence) and comparing them with that from developmental leaf senescence, we were able to show that at early stages of treatments, different hormones and stresses showed limited similarity in the induction of gene expression to that of developmental leaf senescence. Once the senescence process is initiated, as evidenced by visible yellowing, generally after a prolonged period of treatments, a great proportion of SAGs of developmental leaf senescence are shared by gene expression profiles in response to different treatments. This indicates that although different signals that lead to initiation of senescence may do so through distinct signal transduction pathways, senescence processes induced either developmentally or by different senescence-promoting treatments may share common execution events.

Arabidopsis AtNAP regulates fruit senescence.

Arabidopsis has been used as a model system to study many aspects of plant growth and development. However, fruit senescence in Arabidopsis has been less investigated and the underlying molecular and hormonal (especially ethylene) regulatory mechanisms are not well understood. It is reported here that the Arabidopsis silique has characteristics of a climacteric fruit, and that AtNAP, a NAC family transcription factor gene whose expression is increased with the progression of silique senescence, plays an important role in its senescence. Silique senescence was delayed for 4-5 d in the atnap knockout mutant plants. The ethylene climacteric was delayed for 2 d in the atnap silique and the associated respiratory climacteric was suppressed. Exogenous ethylene stimulated respiration in the wild type, but not in the atnap mutant. The decoupling of the ethylene and respiratory climacterics in the atnap mutant suggests that AtNAP is required for ethylene stimulation of respiration. qPCR analyses revealed that the expression patterns of genes involved in ethylene biosynthesis, perception, and signalling, ACS2, ETR1, CTR1, EIN2, EIN3, and ERF1, were also altered in the atnap mutant. The effects of exogenous ABA, SA, 6-BA, and NAA on ethylene production and respiration in siliques of the wild type and atnap mutant were also investigated. A model involving ABA-AtNAP-controlled stomatal opening in regulating ethylene-stimulated respiration in fruit senescence is presented.

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 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.

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.