ARF2-PIF5 interaction controls transcriptional reprogramming in the ABS3-mediated plant senescence pathway.

One of the hallmarks of plant senescence is the global transcriptional reprogramming coordinated by a plethora of transcription factors (TFs). However, mechanisms underlying the interactions between different TFs in modulating senescence remain obscure. Previously, we discovered that plant ABS3 subfamily MATE transporter genes regulate senescence and senescence-associated transcriptional changes. In a genetic screen for mutants suppressing the accelerated senescence phenotype of the gain-of-function mutant abs3-1D, AUXIN RESPONSE FACTOR 2 (ARF2) and PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) were identified as key TFs responsible for transcriptional regulation in the ABS3-mediated senescence pathway. ARF2 and PIF5 (as well as PIF4) interact directly and function interdependently to promote senescence, and they share common target genes such as key senescence promoting genes ORESARA 1 (ORE1) and STAY-GREEN 1 (SGR1) in the ABS3-mediated senescence pathway. In addition, we discovered reciprocal regulation between ABS3-subfamily MATEs and the ARF2 and PIF5/4 TFs. Taken together, our findings reveal a regulatory paradigm in which the ARF2-PIF5/4 functional module facilitates the transcriptional reprogramming in the ABS3-mediated senescence pathway.

The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures.

Light and temperature are two core environmental factors that coordinately regulate plant growth and survival throughout their entire life cycle. However, the mechanisms integrating light and temperature signaling pathways in plants remain poorly understood. Here, we report that CBF1, an AP2/ERF-family transcription factor essential for plant cold acclimation, promotes hypocotyl growth under ambient temperatures in Arabidopsis. We show that CBF1 increases the protein abundance of PIF4 and PIF5, two phytochrome-interacting bHLH-family transcription factors that play pivotal roles in modulating plant growth and development, by directly binding to their promoters to induce their gene expression, and by inhibiting their interaction with phyB in the light. Moreover, our data demonstrate that CBF1 promotes PIF4/PIF5 protein accumulation and hypocotyl growth at both 22°C and 17°C, but not at 4°C, with a more prominent role at 17°C than at 22°C. Together, our study reveals that CBF1 integrates light and temperature control of hypocotyl growth by promoting PIF4 and PIF5 protein abundance in the light, thus providing insights into the integration mechanisms of light and temperature signaling pathways in plants.

AC81 Is a Putative Disulfide Isomerase Involved in Baculoviral Disulfide Bond Formation.

The correct formation of native disulfide bonds is critical for the proper structure and function of many proteins. Cellular disulfide bond formation pathways commonly consist of two parts: sulfhydryl oxidase-mediated oxidation and disulfide isomerase-mediated isomerization. Some large DNA viruses, such as baculoviruses, encode sulfhydryl oxidases, but viral disulfide isomerases have not yet been identified, although G4L in poxvirus has been suggested to serve such a function. Here, we report that the baculovirus core gene ac81 encodes a putative disulfide isomerase. ac81 is conserved in baculoviruses, nudiviruses, and hytrosaviruses. We found that AC81 homologs contain a typical thioredoxin fold conserved in disulfide isomerases. To determine the role of AC81, a series of Autographa californica nucleopolyhedrovirus (AcMNPV) bacmids containing ac81 knockout or point mutations was generated, and the results showed that AC81 is essential for budded virus production, multinucleocapsid occlusion-derived virus (ODV) formation, and ODV embedding in occlusion bodies. Nonreducing Western blot analysis indicated that disulfide bond formation in per os infectivity factor 5 (PIF5), a substrate of the baculoviral sulfhydryl oxidase P33, was abnormal when ac81 was knocked out or mutated. Pulldown assays showed that AC81 interacted with PIF5 and P33 in infected cells. In addition, two critical regions that harbor key amino acids for function were identified in AC81. Taken together, our results suggest that AC81 is a key component involved in the baculovirus disulfide bond formation pathway and likely functions as a disulfide isomerase. IMPORTANCE Many large DNA viruses, such as poxvirus, asfarvirus, and baculovirus, encode their own sulfhydryl oxidase to facilitate the disulfide bond formation of viral proteins. Here, we show that AC81 functions as a putative disulfide isomerase and is involved in multiple functions of the baculovirus life cycle. Interestingly, AC81 and P33 (sulfhydryl oxidase) are conserved in baculoviruses, nudiviruses, and hytrosaviruses, which are all insect-specific large DNA viruses replicating in the nucleus, suggesting that viral disulfide bond formation is an ancient mechanism shared by these viruses.

Structural Characterization of Per Os Infectivity Factor 5 (PIF5) Reveals the Essential Role of Intramolecular Interactions in Baculoviral Oral Infectivity.

Baculoviruses initiate oral infection in the highly alkaline midgut of insects via a group of envelope proteins called per os infectivity factors (PIFs). To date, no high-resolution structural information has been reported for any PIF. Here, we present the crystal structure of the PIF5 ectodomain (PIF5e) from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) at a 2.2-Å resolution. It revealed an open cavity between the N-terminal E1 domain and the C-terminal E2 domain and a cysteine-rich region with three pairs of disulfide bonds in the E2 domain. Multiple conserved intramolecular interactions within PIF5 are essential for maintaining its tertiary structure. Two conserved arginines (Arg8 and Arg74) play critical roles in E1-E2 interactions, and mutagenesis analysis supported their crucial role in oral infection. Importantly, the reduction in the oral infectivity of the Arg8, Arg74, or cysteine mutant viruses was related to the proteolytic cleavage of PIF5 by the endogenous protease embedded in occlusion bodies during alkaline treatment. This suggested that the structural stability of PIF5 under physiological conditions in the insect midgut is critical for baculoviral oral infectivity. IMPORTANCEPer os infection mediated by PIFs is the highly complex mechanism by which baculoviruses initiate infection in insects. Previous studies revealed that multiple PIF proteins form a large PIF complex on the envelope of virions, while PIF5 functions independently of the PIF complex. Here, we report the crystal structure of AcMNPV PIF5e, which, to our knowledge, is the first atomic structure reported for a PIF protein. The structure revealed the precise locations of three previously proposed disulfide bonds and other conserved intramolecular interactions, which are important for the structural stability of PIF5 and are also essential for oral infectivity. These findings advance our understanding of the molecular mechanism of baculovirus oral infection under alkaline conditions.

COP1 SUPPRESSOR 6 represses the PIF4 and PIF5 action to promote light-inhibited hypocotyl growth.

Light signaling precisely controls photomorphogenic development in plants. PHYTOCHROME INTERACTING FACTOR 4 and 5 (PIF4 and PIF5) play critical roles in the regulation of this developmental process. In this study, we report CONSTITUTIVELY PHOTOMORPHOGENIC 1 SUPPRESSOR 6 (CSU6) functions as a key regulator of light signaling. Loss of CSU6 function largely rescues the cop1-6 constitutively photomorphogenic phenotype. CSU6 promotes hypocotyl growth in the dark, but inhibits hypocotyl elongation in the light. CSU6 not only associates with the promoter regions of PIF4 and PIF5 to inhibit their expression in the morning, but also directly interacts with both PIF4 and PIF5 to repress their transcriptional activation activity. CSU6 negatively controls a group of PIF4- and PIF5-regulated gene expressions. Mutations in PIF4 and/or PIF5 are epistatic to the loss of CSU6, suggesting that CSU6 acts upstream of PIF4 and PIF5. Taken together, CSU6 promotes light-inhibited hypocotyl elongation by negatively regulating PIF4 and PIF5 transcription and biochemical activity.

Degradation of the transcription factors PIF4 and PIF5 under UV-B promotes UVR8-mediated inhibition of hypocotyl growth in Arabidopsis.

Inhibition of hypocotyl growth is a well-established UV-B-induced photomorphogenic response that is mediated by the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8). However, the molecular mechanism by which UVR8 signaling triggers inhibition of hypocotyl growth is poorly understood. The bZIP protein ELONGATED HYPOCOTYL 5 (HY5) functions as the main positive regulatory transcription factor in the UVR8 signaling pathway, with HY5-HOMOLOG (HYH) playing a minor role. However, here we demonstrate that hy5 hyh double mutants maintain significant UVR8-dependent hypocotyl growth inhibition. We identify UVR8-dependent inhibition of the activities of bHLH transcription factors PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5 as part of the UVR8 signaling pathway, which results in inhibition of hypocotyl growth. The UVR8-mediated repression of several hypocotyl elongation-related genes is independent of HY5 and HYH but largely associated with UVR8-dependent degradation of PIF4 and PIF5, a process that consequently diminishes PIF4/5 target promoter occupancy. Taken together, our data indicate that UVR8-mediated inhibition of hypocotyl growth involves degradation of PIF4 and PIF5. These findings contribute to our mechanistic understanding of UVR8-induced photomorphogenesis and further support the function of PIFs as integrators of different photoreceptor signaling pathways.

Per Os Infectivity Factor 5 Identified as a Substrate of P33 in the Baculoviral Disulfide Bond Formation Pathway.

Disulfide bonds are critical for the structure and function of many proteins. Some large DNA viruses encode their own sulfhydryl oxidase for disulfide bond formation. Previous studies have demonstrated that the baculovirus-encoded sulfhydryl oxidase P33 is necessary for progeny virus production, and its enzymatic activity is important for morphogenesis and oral infectivity of baculoviruses. However, the downstream substrates of P33 in the putative redox pathway of baculoviruses are unknown. In this study, we showed that PIF5, one of the per os infectivity factors (PIFs), contained intramolecular disulfide bonds and that the disulfide bond formation was interrupted in the absence of P33. In vivo pulldown and colocalization analyses revealed that PIF5 and P33 interacted with each other during virus infection. Further, in vitro assays validated that the reduced PIF5 proteins could be oxidized by P33. To understand the contribution of disulfide bonds to the function of PIF5, several cysteine-to-serine mutants were constructed, which all interfered with the disulfide bond formation of PIF5 to different extents. All the mutants lost their oral infectivity but had no impact on infectious budding virus (BV) production or virus morphogenesis. Taken together, our results indicated PIF5 as the first identified substrate of P33. Further, the disulfide bonds in PIF5 play an essential role in its function in oral infection.IMPORTANCE Similar to some large DNA viruses that encode their own disulfide bond pathway, baculovirus encodes a viral sulfhydryl oxidase, P33. Enzyme activity of P33 is related to infectious BV production, occlusion-derived virus (ODV) envelopment, occlusion body morphogenesis, and oral infectivity, suggesting that P33 is involved in disulfide bond formation of multiple proteins. A complete disulfide bond formation pathway normally contains a sulfhydryl oxidase, a disulfide-donating enzyme, and one or more substrates. In baculovirus, apart from P33, other components of the putative pathway remain unknown. In this study, we identified PIF5 as the first substrate of P33, which is fundamental for revealing the complete disulfide bond formation pathway in baculovirus. PIF5 is essential for oral infection and is absent from the PIF complex. Our study demonstrated that native disulfide bonds in PIF5 are required for oral infection, which will help us to reveal its mode of action.

PHYTOCHROME INTERACTING FACTORS PIF4 and PIF5 promote heat stress induced leaf senescence in Arabidopsis.

Leaf senescence can be triggered by multiple abiotic stresses including darkness, nutrient limitation, salinity, and drought. Recently, heatwaves have been occurring more frequently, and they dramatically affect plant growth and development. However, the underlying molecular networks of heat stress-induced leaf senescence remain largely uncharacterized. Here we showed that PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5 proteins could efficiently promote heat stress-induced leaf senescence in Arabidopsis. Transcriptomic profiling analysis revealed that PIF4 and PIF5 are likely to function through multiple biological processes including hormone signaling pathways. Further, we characterized NAC019, SAG113, and IAA29 as direct transcriptional targets of PIF4 and PIF5. The transcription of NAC019, SAG113, and IAA29 changes significantly in daytime after heat treatment. In addition, we demonstrated that PIF4 and PIF5 proteins were accumulated during the recovery after heat treatment. Moreover, we showed that heat stress-induced leaf senescence is gated by the circadian clock, and plants might be more actively responsive to heat stress-induced senescence during the day. Taken together, our findings proposed important roles for PIF4 and PIF5 in mediating heat stress-induced leaf senescence, which may help to fully illustrate the molecular network of heat stress-induced leaf senescence in higher plants and facilitate the generation of heat stress-tolerant crops.

Coordinated transcriptional regulation of isopentenyl diphosphate biosynthetic pathway enzymes in plastids by phytochrome-interacting factor 5.

All isoprenoids are derived from a common C5 unit, isopentenyl diphosphate (IPP). In plants, IPP is synthesized via two distinct pathways; the cytosolic mevalonate pathway and the plastidial non-mevalonate (MEP) pathway. In this study, we used a co-expression analysis to identify transcription factors that coordinately regulate the expression of multiple genes encoding enzymes in the IPP biosynthetic pathway. Some candidates showed especially strong correlations with multiple genes encoding MEP-pathway enzymes. We report here that phytochrome-interacting factor 5 (PIF5), a basic-helix-loop-helix type transcription factor, functions as a positive regulator of the MEP pathway. Its overexpression in T87 suspension cultured cells resulted in increased accumulation of chlorophylls and carotenoids. Detailed analyses of carotenoids by HPLC indicated that some carotenoid biosynthetic pathways were concomitantly up-regulated, possibly as a result of enhanced IPP metabolic flow. Our results also revealed other PIF family proteins that play different roles from that of PIF5 in IPP metabolism.

The time of day effects of warm temperature on flowering time involve PIF4 and PIF5.

Warm temperature promotes flowering in Arabidopsis thaliana and this response involves multiple signalling pathways. To understand the temporal dynamics of temperature perception, tests were carried out to determine if there was a daily window of enhanced sensitivity to warm temperature (28 °C). Warm temperature applied during daytime, night-time, or continuously elicited earlier flowering, but the effects of each treatment were unequal. Plants exposed to warm night (WN) conditions flowered nearly as early as those in constant warm (CW) conditions, while treatment with warm days (WD) caused later flowering than either WN or CW. Flowering in each condition relied to varying degrees on the activity of CO , FT , PIF4 , and PIF5 , as well as the action of unknown genes. The combination of signalling pathways involved in flowering depended on the time of the temperature cue. WN treatments caused a significant advance in the rhythmic expression waveform of CO, which correlated with pronounced up-regulation of FT expression, while WD caused limited changes in CO expression and no stimulation of FT expression. WN- and WD-induced flowering was partially CO independent and, unexpectedly, dependent on PIF4 and PIF5 . pif4-2, pif5-3, and pif4-2 pif5-3 mutants had delayed flowering under all three warm conditions. The double mutant was also late flowering in control conditions. In addition, WN conditions alone imposed selective changes to PIF4 and PIF5 expression. Thus, the PIF4 and PIF5 transcription factors promote flowering by at least two means: inducing FT expression in WN and acting outside of FT by an unknown mechanism in WD.

Non-transcriptional regulatory activity of SMAX1 and SMXL2 mediates karrikin-regulated seedling response to red light in Arabidopsis.

Karrikins and strigolactones govern plant development and environmental responses through closely related signaling pathways. The transcriptional repressor proteins SUPPRESSOR OF MAX2 1 (SMAX1), SMAX1-like2 (SMXL2), and D53-like SMXLs mediate karrikin and strigolactone signaling by directly binding downstream genes or by inhibiting the activities of transcription factors. In this study, we characterized the non-transcriptional regulatory activities of SMXL proteins in Arabidopsis. We discovered that SMAX1 and SMXL2 with mutations in their ethylene-response factor-associated amphiphilic repression (EAR) motif had undetectable or weak transcriptional repression activities but still partially rescued the hypocotyl elongation defects and fully reversed the cotyledon epinasty defects of the smax1 smxl2 mutant. SMAX1 and SMXL2 directly interact with PHYTOCHROME INTERACTION FACTOR 4 (PIF4) and PIF5 to enhance their protein stability by interacting with phytochrome B (phyB) and suppressing the association of phyB with PIF4 and PIF5. The karrikin-responsive genes were then identified by treatment with GR24ent-5DS, a GR24 analog showing karrikin activity. Interestingly, INDOLE-3-ACETIC ACID INDUCIBLE 29 (IAA29) expression was repressed by GR24ent-5DS treatment in a PIF4- and PIF5-dependent and EAR-independent manner, whereas KARRIKIN UPREGULATED F-BOX 1 (KUF1) expression was induced in a PIF4- and PIF5-independent and EAR-dependent manner. Furthermore, the non-transcriptional regulatory activity of SMAX1, which is independent of the EAR motif, had a global effect on gene expression. Taken together, these results indicate that non-transcriptional regulatory activities of SMAX1 and SMXL2 mediate karrikin-regulated seedling response to red light.

A molecular framework underlying low-nitrogen-induced early leaf senescence in Arabidopsis thaliana.

Nitrogen (N) deficiency causes early leaf senescence, resulting in accelerated whole-plant maturation and severely reduced crop yield. However, the molecular mechanisms underlying N-deficiency-induced early leaf senescence remain unclear, even in the model species Arabidopsis thaliana. In this study, we identified Growth, Development and Splicing 1 (GDS1), a previously reported transcription factor, as a new regulator of nitrate (NO3-) signaling by a yeast-one-hybrid screen using a NO3- enhancer fragment from the promoter of NRT2.1. We showed that GDS1 promotes NO3- signaling, absorption and assimilation by affecting the expression of multiple NO3- regulatory genes, including Nitrate Regulatory Gene2 (NRG2). Interestingly, we observed that gds1 mutants show early leaf senescence as well as reduced NO3- content and N uptake under N-deficient conditions. Further analyses indicated that GDS1 binds to the promoters of several senescence-related genes, including Phytochrome-Interacting Transcription Factors 4 and 5 (PIF4 and PIF5) and represses their expression. Interestingly, we found that N deficiency decreases GDS1 protein accumulation, and GDS1 could interact with Anaphase Promoting Complex Subunit 10 (APC10). Genetic and biochemical experiments demonstrated that Anaphase Promoting Complex or Cyclosome (APC/C) promotes the ubiquitination and degradation of GDS1 under N deficiency, resulting in loss of PIF4 and PIF5 repression and consequent early leaf senescence. Furthermore, we discovered that overexpression of GDS1 could delay leaf senescence and improve seed yield and N-use efficiency (NUE) in Arabidopsis. In summary, our study uncovers a molecular framework illustrating a new mechanism underlying low-N-induced early leaf senescence and provides potential targets for genetic improvement of crop varieties with increased yield and NUE.

Arabidopsis FHY3 and FAR1 Function in Age Gating of Leaf Senescence.

Leaf senescence is the terminal stage of leaf development. Both light and the plant hormone ethylene play important roles in regulating leaf senescence. However, how they coordinately regulate leaf senescence during leaf development remains largely unclear. In this study, we show that FHY3 and FAR1, two homologous proteins essential for phytochrome A-mediated light signaling, physically interact with and repress the DNA binding activity of EIN3 (a key transcription factor essential for ethylene signaling) and PIF5 (a bHLH transcription factor negatively regulating light signaling), and interfere with their DNA binding to the promoter of ORE1, which encodes a key NAC transcription factor promoting leaf senescence. In addition, we show that FHY3, PIF5, and EIN3 form a tri-protein complex(es) and that they coordinately regulate the progression of leaf senescence. We show that during aging or under dark conditions, accumulation of FHY3 protein decreases, thus lifting its repression on DNA binding of EIN3 and PIF5, leading to the increase of ORE1 expression and onset of leaf senescence. Our combined results suggest that FHY3 and FAR1 act in an age gating mechanism to prevent precocious leaf senescence by integrating light and ethylene signaling with developmental aging.