MPK4 phosphorylates MYC2 transcription factors to regulate jasmonic acid signaling and herbivory responses in maize.

Regulation of responses induced by herbivory and jasmonic acid (JA) remains poorly understood in the important staple crop maize (Zea mays). MYC2 is the key transcription factor regulating many aspects of JA signaling, while mitogen-activated protein kinases (MAPKs or MPKs) play important roles in various plant physiological processes. Using a combination of reverse genetics, transcriptome analysis, and biochemical assays, we elucidated the important role of MPK4 in maize resistance to insects and in JA signaling. Silencing MPK4 increased the JA and jasmonoyl-isoleucine levels elicited by wounding or simulated herbivory but decreased maize resistance to armyworm (Mythimna separata) larvae. We showed that MPK4 is required for transcriptional regulation of many genes responsive to methyl jasmonate, indicating the important role of maize MPK4 in JA signaling. Biochemical analyses indicated that MPK4 directly phosphorylates MYC2s at Thr115 of MYC2a and Thr112 of MYC2b. Compared with nonphosphorylated MYC2s, phosphorylated MYC2s were more prone to degradation and exhibited enhanced transactivation activity against the promoters of several benzoxazinoid biosynthesis genes, which are important for maize defense against insects. This study reveals the essential role of maize MPK4 in JA signaling and provides insights into the functions of MAPKs in maize.

The TOR complex controls ATP levels to regulate actin cytoskeleton dynamics in Arabidopsis.

Energy is essential for all cellular functions in a living organism. How cells coordinate their physiological processes with energy status and availability is thus an important question. The turnover of actin cytoskeleton between its monomeric and filamentous forms is a major energy drain in eukaryotic cells. However, how actin dynamics are regulated by ATP levels remain largely unknown in plant cells. Here, we observed that seedlings with impaired functions of target of rapamycin complex 1 (TORC1), either by mutation of the key component, RAPTOR1B, or inhibition of TOR activity by specific inhibitors, displayed reduced sensitivity to actin cytoskeleton disruptors compared to their controls. Consistently, actin filament dynamics, but not organization, were suppressed in TORC1-impaired cells. Subcellular localization analysis and quantification of ATP concentration demonstrated that RAPTOR1B localized at cytoplasm and mitochondria and that ATP levels were significantly reduced in TORC1-impaired plants. Further pharmacologic experiments showed that the inhibition of mitochondrial functions led to phenotypes mimicking those observed in raptor1b mutants at the level of both plant growth and actin dynamics. Exogenous feeding of adenine could partially restore ATP levels and actin dynamics in TORC1-deficient plants. Thus, these data support an important role for TORC1 in coordinating ATP homeostasis and actin dynamics in plant cells.

Dodder-transmitted mobile signals prime host plants for enhanced salt tolerance.

The dodders (Cuscuta spp.) are a genus of shoot parasites. In nature, a dodder often simultaneously parasitizes two or more neighboring hosts. Salt stress is a common abiotic stress for plants. It is unclear whether dodder transmits physiologically relevant salt stress-induced systemic signals among its hosts and whether these systemic signals affect the hosts' tolerance to salt stress. Here, we simultaneously parasitized two or more cucumber plants with dodder. We found that salt treatment of one host highly primed the connected host, which showed strong decreases in the extent of leaf withering and cell death in response to subsequent salt stress. Transcriptomic analysis indicated that 24 h after salt treatment of one cucumber, the transcriptome of the other dodder-connected cucumber largely resembled that of the salt-treated one, indicating that inter-plant systemic signals primed these dodder-connected cucumbers at least partly through transcriptomic reconfiguration. Furthermore, salt treatment of one of the cucumbers induced physiological changes, including altered proline contents, stomatal conductance, and photosynthetic rates, in both of the dodder-connected cucumbers. This study reveals a role of dodder in mediating salt-induced inter-plant signaling among dodder-connected hosts and highlights the physiological function of these mobile signals in plant-plant interactions under salt stress.

The host jasmonic acid pathway regulates the transcriptomic changes of dodder and host plant under the scenario of caterpillar feeding on dodder.

BACKGROUND: Dodder (Cuscuta spp., Convolvulaceae) species are obligate leaf- and rootless parasites that totally depend on hosts to survive. Dodders naturally graft themselves to host stems to form vascular fusion, from which they obtain nutrients and water. In addition, dodders and their hosts also exchange various other molecules, including proteins, mRNAs, and small RNAs. It is very likely that vascular fusion also allows inter-plant translocation of systemic signals between dodders and host plants and these systemic signals may have profound impacts on the physiology of dodder and host plants. Herbivory is a common biotic stress for plants. When a dodder parasite is attacked by lepidopteran insects, how dodder responds to caterpillar feeding and whether there are inter-plant communications between the host plants and the parasites is still poorly understood. RESULTS: Here, wild-type (WT) tobacco and a tobacco line in which jasmonic acid (JA) biosynthesis was silenced (AOC-RNAi) were used as the hosts, and the responses of dodders and their host plants to herbivory by Spodoptera litura caterpillars on the dodders were investigated. It was found that after caterpillar attack, dodders grown on AOC-RNAi tobacco showed much a smaller number of differentially expressed genes, although the genotypes of the tobacco plants did not have an effect on the simulated S. litura feeding-induced JA accumulation in dodders. We further show that S. litura herbivory on dodder also led to large changes in transcriptome and defensive metabolites in the host tobacco, leading to enhanced resistance to S. litura, and the JA pathway of tobacco host is critical for these systemic responses. CONCLUSIONS: Our findings indicate that during caterpillar attack on dodder, the JA pathway of host plant is required for the proper transcriptomic responses of both dodder and host plants. This study highlights the importance of the host JA pathway in regulating the inter-plant systemic signaling between dodder and hosts.

Large-scale interplant exchange of macromolecules between soybean and dodder under nutrient stresses.

Parasitic plants and their hosts communicate through haustorial connections. Nutrient deficiency is a common stress for plants, yet little is known about whether and how host plants and parasites communicate during adaptation to such nutrient stresses. In this study, we used transcriptomics and proteomics to analyze how soybean (Glycine max) and its parasitizing dodder (Cuscuta australis) respond to nitrate and phosphate deficiency (-N and -P). After -N and -P treatment, the soybean and dodder plants exhibited substantial changes of transcriptome and proteome, although soybean plants showed very few transcriptional responses to -P and dodder did not show any transcriptional changes to either -N or -P. Importantly, large-scale interplant transport of mRNAs and proteins was detected. Although the mobile mRNAs only comprised at most 0.2% of the transcriptomes, the foreign mobile proteins could reach 6.8% of the total proteins, suggesting that proteins may be the major forms of interplant communications. Furthermore, the interplant mobility of macromolecules was specifically affected by the nutrient regimes and the transport of these macromolecules was very likely independently regulated. This study provides new insight into the communication between host plants and parasites under stress conditions.

Transceptor NRT1.1 and receptor-kinase QSK1 complex controls PM H+-ATPase activity under low nitrate.

NRT1.1, a nitrate transceptor, plays an important role in nitrate binding, sensing, and nitrate-dependent lateral root (LR) morphology. However, little is known about NRT1.1-mediated nitrate signaling transduction through plasma membrane (PM)-localized proteins. Through in-depth phosphoproteome profiling using membranes of Arabidopsis roots, we identified receptor kinase QSK1 and plasma membrane H+-ATPase AHA2 as potential downstream components of NRT1.1 signaling in a mild low-nitrate (LN)-dependent manner. QSK1, as a functional kinase and molecular link, physically interacts with NRT1.1 and AHA2 at LN and specifically phosphorylates AHA2 at S899. Importantly, we found that LN, not high nitrate (HN), induces formation of the NRT1.1-QSK1-AHA2 complex in order to repress the proton efflux into the apoplast by increased phosphorylation of AHA2 at S899. Loss of either NRT1.1 or QSK1 thus results in a higher T947/S899 phosphorylation ratio on AHA2, leading to enhanced pump activity and longer LRs under LN. Our results uncover a regulatory mechanism in which NRT1.1, under LN conditions, promotes coreceptor QSK1 phosphorylation and enhances the NRT1.1-QSK1 complex formation to transduce LN sensing to the PM H+-ATPase AHA2, controlling the phosphorylation ratio of activating and inhibitory phosphorylation sites on AHA2. This then results in altered proton pump activity, apoplast acidification, and regulation of NRT1.1-mediated LR growth.

Multiomics Analysis Reveals the Chemical and Genetic Bases of Pigmented Potato Tuber.

Anthocyanins and carotenoids determine the diversity of potato tuber flesh pigmentation; here, the underlying chemical and genetic bases were elucidated by multiomics analyses. A total of 31 anthocyanins and 30 carotenoids were quantified in five differently pigmented tubers. Cyanidin and pelargonidin derivatives determined the redness, while malvidin, petunidin, and delphinidin derivatives contributed to purpleness. Violaxanthin derivatives determined the light-yellow color, while zeaxanthin and antheraxanthin derivatives further enhanced the deep-yellow deposition. Integrated transcriptome and proteome analyses identified that F3'5'H highly enhanced anthocyanin biosynthesis in purple flesh and was responsible for metabolic divergence between red and purple samples. BCH2 significantly enhanced carotenoid biosynthesis in yellow samples and along with ZEP, NCED1, and CCD1 genes determined metabolic divergence between light and deep-yellow samples. The weighted correlation network analysis constructed a regulatory network revealing the central role of AN1 in regulating anthocyanin biosynthesis, and 10 new transcription factors related to anthocyanin and carotenoid metabolism regulation were identified. Our findings provide targeted genes controlling tuber pigmentation, which will be meaningful for the genetic manipulation of tuber quality improvement.

Phosphoproteomics Analysis of Plant Root Tissue.

Plants absorb water and nutrients from soil through roots and transmit these resources through the xylem to the shoot. Roots therefore participate in information and material transduction as well as signal communication with the shoot. The importance of reversible protein phosphorylation in the regulation of plant growth and development has been amply demonstrated through decades of research. Here, we present a simple mass spectrometry-based shotgun phosphoproteomics protocol for Arabidopsis root tissue. Through this method, we can profile the Arabidopsis root phosphoproteome and construct signal networks of key proteins to better understand their roles in plant growth and development.

Plant Phosphopeptide Identification and Label-Free Quantification by MaxQuant and Proteome Discoverer Software.

Both the phosphorylation and dephosphorylation of plant proteins is involved in multiple biological processes, especially in regard to signal transduction. The identification of phosphopeptides from MS (mass spectrometry)-based methods and their subsequent quantification play an important role in plant phosphoproteomics analysis. Phosphopeptide(s) identification and label-free quantification can determine dynamic changes of phosphorylation events in plants. Both MaxQuant and Proteome Discoverer are professional software tools used to identify and quantify large-scale MS-based phosphoproteomic data. This chapter gives a detailed workflow of MaxQuant and Proteome Discoverer software to analyze large amounts of phosphoproteomic-related MS data for the identification and quantification of label-free plant phosphopeptides.

Extensive Inter-plant Protein Transfer between Cuscuta Parasites and Their Host Plants.

Cuscuta species (dodders) are holoparasites that totally rely on host plants to survive. Although various mobile proteins have been identified to travel within a plant, whether and to what extent protein transfer between Cuscuta and host plants remain unclear. We found that hundreds to more than 1500 proteins were transferred between Cuscuta and the host plants Arabidopsis and soybean, and hundreds of inter-plant mobile proteins were even detected in the seeds of Cuscuta and the host soybean. Different hosts bridge-connected by dodder were also found to exchange hundreds of proteins. Quantitatively, the mobile proteins represent a few to more than 10% of the proteomes of foreign plants. Using Arabidopsis plants expressing different reporter proteins, we further showed that these reporter proteins could travel between plants and, importantly, retained their activity in the foreign plants. Comparative analysis between the inter-plant mobile proteins and mRNAs indicated that the majority of mobile proteins were not de novo synthesized from the translocated mRNAs, but bona fide mobile proteins. We propose that large-scale inter-plant protein translocation may play an important role in the interactions between host plants and dodder and even among the dodder bridge-connected hosts.