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Dodder (Cuscuta spp., Convolvulaceae) is a genus of parasitic plants with worldwide distribution. Dodders are able to simultaneously parasitize two or more adjacent hosts, forming dodder-connected plant clusters. Nitrogen (N) deficiency is a common challenge to plants. To date, it has been unclear whether dodder transfers N-systemic signals between hosts grown in N-heterogeneous soil. Transcriptome and methylome analyses were carried out to investigate whether dodder (Cuscuta campestris) transfers N-systemic signals between N-replete and N-depleted cucumber (Cucumis sativus) hosts, and it was found that N-systemic signals from the N-deficient cucumber plants were rapidly translocated through C. campestris to the N-replete cucumber plants. Unexpectedly, certain systemic signals were also transferred from the N-replete to N-depleted cucumber hosts. We demonstrate that these systemic signals are able to regulate large transcriptome and DNA methylome changes in the recipient hosts. Importantly, N stress also induced many long-distance mobile mRNA transfers between C. campestris and hosts, and the bilateral N-systemic signaling between N-replete and N-depleted hosts had a strong impact on the inter-plant mobile mRNAs. Our 15N labeling experiment indicated that under N-heterogeneous conditions, N-systemic signals from the N-deficient cucumber hosts did not obviously change the N-uptake activity of the N-replete cucumber hosts; however, in plant clusters comprising C. campestris-connected cucumber and soybean (Glycine max) plants, if the soybean plants were N-starved, the cucumber plants exhibited increased N-uptake activity. This study reveals that C. campestris facilitates plant–plant communications under N-stress conditions by enabling extensive bilateral N-systemic signaling between different hosts.

Genetic variation within and among populations provides the raw material for evolution. Although many studies describe inter- and intraspecific variation of defensive metabolites, little is known about variation among plant populations within early signaling responses elicited by herbivory or by herbivore oral secretions (OS) introduced into wounds during feeding. In this study, we compare the OS-elicited early responses as well as the antiherbivore defensive metabolites in two accessions of the wild tobacco Nicotiana attenuata and show that, compared with an accession collected from Utah, an Arizona accession has lower herbivore-elicited activity of the salicylic acid-induced protein kinase, an important mitogen-activated protein kinase involved in herbivore resistance. These differences in salicylic acid-induced protein kinase activity were associated with substantially different levels of OS-elicited jasmonic acid, jasmonic acid-isoleucine conjugate, and ethylene bursts. Gene expression level polymorphism (ELP) determines phenotypic variation among populations, and we found the two accessions to have significantly different ELPs in the genes involved in early signaling responses to herbivory. In addition, we found differences between the Utah and the Arizona accessions in the concentrations of several secondary metabolites that contribute to N. attenuata's direct and indirect defenses. This study demonstrates significant natural variation in regulatory elements that mediate plant responses to herbivore attack, highlighting the role of ELP in producing a diversity of plant defense phenotypes.

The plant hormone jasmonic acid (JA) plays a pivotal role in plant-insect interactions. Herbivore attack usually elicits dramatic increases in JA concentrations, which in turn activate the accumulation of metabolites that function as defenses against herbivores. Although almost all enzymes involved in the biosynthesis pathway of JA have been identified and characterized, the mechanism by which plants regulate JA biosynthesis remains unclear. Calcium-dependent protein kinases (CDPKs) are plant-specific proteins that sense changes in [Ca2+] to activate downstream responses. We created transgenic Nicotiana attenuata plants, in which two CDPKs, NaCDPK4 and NaCDPK5, were simultaneously silenced (IRcdpk4/5 plants). IRcdpk4/5 plants were stunted and aborted most of their flower primordia. Importantly, after wounding or simulated herbivory, IRcdpk4/5 plants accumulated exceptionally high JA levels. When NaCDPK4 and NaCDPK5 were silenced individually, neither stunted growth nor high JA levels were observed, suggesting that NaCDPK4 and NaCDPK5 have redundant roles. Attack from Manduca sexta larvae on IRcdpk4/5 plants induced high levels of defense metabolites that slowed M. sexta growth. We found that NaCDPK4 and NaCDPK5 affect plant resistance against insects in a JA- and JA-signaling-dependent manner. Furthermore, IRcdpk4/5 plants showed overactivation of salicylic-acid-induced protein kinase, a mitogen-activated protein kinase involved in various stress responses, and genetic analysis indicated that the increased salicylic-acid-induced protein kinase activity in IRcdpk4/5 plants was a consequence of the exceptionally high JA levels and was dependent on CORONATINE INSENSITIVE1. This work reveals the critical roles of CDPKs in modulating JA homeostasis and highlights the complex duet between JA and mitogen-activated protein kinase signaling.

Mitogen-activated protein kinases (MAPKs) play pivotal roles in development and environmental interactions in eukaryotes. Here, we studied the function of a MAPK, NaMPK4, in the wild tobacco species Nicotiana attenuata. The NaMPK4-silenced N. attenuata (irNaMPK4) attained somewhat smaller stature, delayed senescence, and greatly enhanced stomatal conductance and photosynthetic rate, especially during late developmental stages. All these changes were associated with highly increased seed production. Using leaf epidermal peels, we demonstrate that guard cell closure in irNaMPK4 was strongly impaired in response to abscisic acid and hydrogen peroxide, and consistently, irNaMPK4 plants transpired more water and wilted sooner than did wild-type plants when they were deprived of water. We show that NaMPK4 plays an important role in the guard cell-mediated defense against a surface-deposited bacterial pathogen, Pseudomonas syringae pv tomato (Pst) DC3000; in contrast, when bacteria directly entered leaves by pressure infiltration, NaMPK4 was found to be less important in the resistance to apoplast-located Pst DC3000. Moreover, we show that salicylic acid was not involved in the defense against PstDC3000 in wild-type and irNaMPK4 plants once it had entered leaf tissue. Finally, we provide evidence that NaMPK4 functions differently from AtMPK4 and AtMPK11 in Arabidopsis (Arabidopsis thaliana), despite their sequence similarities, suggesting a complex functional divergence of MAPKs in different plant lineages. This work highlights the multifaceted functions of NaMPK4 in guard cells and underscores its role in mediating various ecologically important traits.

Trypsin proteinase inhibitors (TPIs) of Nicotiana attenuata are major antiherbivore defenses that increase dramatically in leaves after attack or methyl jasmonate (MeJA) elicitation. To understand the elicitation process, we characterized the proteolytic fragmentation and release of TPIs from a multidomain precursor by proteases in MeJA-elicited and unelicited plants. A set of approximately 6-kD TPI peptides was purified from leaves, and their posttranslational modifications were characterized. In MeJA-elicited plants, the diversity of TPI structures was greater than the precursor gene predicted. This elicited structural heterogeneity resulted from differential fragmentation of the linker peptide (LP) that separates the seven-domain TPI functional domains. Using an in vitro fluorescence resonance energy transfer assay and synthetic substrates derived from the LP sequence, we characterized proteases involved in both the processing of the TPI precursor and its vacuolar targeting sequence. Although both a vacuolar processing enzyme and a subtilisin-like protease were found to participate in a two-step processing of LP, only the activity of the subtilisin-like protease was significantly increased by MeJA elicitation. We propose that MeJA elicitation increases TPI precursor production and saturates the proteolytic machinery, changing the processing pattern of TPIs. To test this hypothesis, we elicited a TPI-deficient N. attenuata genotype that had been transformed with a functional NaTPI gene under control of a constitutive promoter and characterized the resulting TPIs. We found no alterations in the processing pattern predicted from the sequence: a result consistent with the saturation hypothesis.