Plants can talk: a new era in plant acoustics.

Plants release chemical signals to interact with their environment when exposed to stress. Khait and colleagues unveiled that plants 'verbalize' stress by emitting airborne sounds. These can train machine learning models to identify plant stressors. This unlocks a new path in plant-environment interactions research with multiple possibilities for future applications.

Sound perception in plants: from ecological significance to molecular understanding.

In addition to positive effects on plant growth and resilience, sound alerts plants of potential danger and aids in defense. Sound guides plants towards essential resources, like water, through phonotropic root growth. Sound also facilitates mutualistic interactions such as buzz pollination. Molecularly, sound induces Ca2+ signatures, K+ fluxes, and an increase in reactive oxygen species (ROS) levels in a mechanosensitive ion channel-dependent fashion. We review the two major open questions in the field of plant acoustics: (i) what is the ecological relevance of sound in plant life, and (ii) how is sound sensed and transduced to evoke a morphophysiological response? We highlight the clear need to combine the ecological and molecular perspectives for a more holistic approach to better understand plant behavior.

Arabidopsis plants overexpressing additional copies of heat shock protein Hsp101 showed high heat tolerance and endo-gene silencing.

Hsp101 chaperone is vital for survival of plants under heat stress. We generated transgenic Arabidopsis thaliana (Arabidopsis) lines with extra copies of Hsp101 gene using diverse approaches. Arabidopsis plants transformed with rice Hsp101 cDNA driven by Arabidopsis Hsp101 promoter (IN lines) showed high heat tolerance while the plants transformed with rice Hsp101 cDNA driven by CaMV35S promoter (C lines) were like wild type plants in heat stress response. Transformation of Col-0 plants with 4633 bp Hsp101 genomic fragment (GF lines) from A. thaliana containing both its coding and the regulatory sequence resulted in mostly over-expressor (OX) lines and a few under-expressor (UX) lines of Hsp101. OX lines showed enhanced heat tolerance while the UX lines were overly heat sensitive. In UX lines, silencing of not only Hsp101 endo-gene was noted but also transcript of choline kinase (CK2) was silenced. Previous work established that in Arabidopsis, CK2 and Hsp101 are convergent gene pairs sharing a bidirectional promoter. The elevated AtHsp101 protein amount in most GF and IN lines was accompanied by lowered CK2 transcript levels under HS. We observed increased methylation of the promoter and gene sequence region in UX lines; however, methylation was lacking in OX lines.

The 5-formyl-tetrahydrofolate proteome links folates with C/N metabolism and reveals feedback regulation of folate biosynthesis.

Folates are indispensable for plant development, but their molecular mode of action remains elusive. We synthesized a probe, "5-F-THF-Dayne," comprising 5-formyl-tetrahydrofolate (THF) coupled to a photoaffinity tag. Exploiting this probe in an affinity proteomics study in Arabidopsis thaliana, we retrieved 51 hits. Thirty interactions were independently validated with in vitro expressed proteins to bind 5-F-THF with high or low affinity. Interestingly, the interactors reveal associations beyond one-carbon metabolism, covering also connections to nitrogen (N) metabolism, carbohydrate metabolism/photosynthesis, and proteostasis. Two of the interactions, one with the folate biosynthetic enzyme DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE 1 (AtDHFR-TS1) and another with N metabolism-associated glutamine synthetase 1;4 (AtGLN1;4), were further characterized. In silico and experimental analyses revealed G35/K36 and E330 as key residues for the binding of 5-F-THF in AtDHFR-TS1 and AtGLN1;4, respectively. Site-directed mutagenesis of AtGLN1;4 E330, which co-localizes with the ATP-binding pocket, abolished 5-F-THF binding as well as AtGLN1;4 activity. Furthermore, 5-F-THF was noted to competitively inhibit the activities of AtDHFR-TS1 and AtGLN1;4. In summary, we demonstrated a regulatory role for 5-F-THF in N metabolism, revealed 5-F-THF-mediated feedback regulation of folate biosynthesis, and identified a total of 14 previously unknown high-affinity binding cellular targets of 5-F-THF. Together, this sets a landmark toward understanding the role of folates in plant development.

Drought-induced susceptibility for Cenangium ferruginosum leads to progression of Cenangium-dieback disease in Pinus koraiensis.

Recently, the occurrence of "Cenangium-dieback" has been frequent and devastating. Cenangium-dieback is caused by an endophytic fungus Cenangium ferruginosum in stressed pine trees. Progression of the disease in terms of molecular interaction between host and pathogen is not well studied and there is a need to develop preventive strategies. Thus, we simulated disease conditions and studied the associated transcriptomics, metabolomics, and hormonal changes. Pinus koraiensis seedlings inoculated with C. ferruginosum were analyzed both under drought and well-watered conditions. Transcriptomic analysis suggested decreased expression of defense-related genes in C. ferruginosum-infected seedlings experiencing water-deficit. Further, metabolomic analysis indicated a decrease in the key antimicrobial terpenoids, flavonoids, and phenolic acids. Hormonal analysis revealed a drought-induced accumulation of abscisic acid and a corresponding decline in the defense-associated jasmonic acid levels. Pathogen-associated changes were also studied by treating C. ferruginosum with metabolic extracts from pine seedlings (with and without drought) and polyethylene glycol to simulate the effects of direct drought. From RNA sequencing and metabolomic analysis it was determined that drought did not directly induce pathogenicity of C. ferruginosum. Collectively, we propose that drought weakens pine immunity, which facilitates increased C. ferruginosum growth and results in conversion of the endophyte into the phytopathogen causing dieback.

Fungal endophytes inhabiting mountain-cultivated ginseng (Panax ginseng Meyer): Diversity and biocontrol activity against ginseng pathogens.

Fungal endophytes isolated from mountain-cultivated ginseng (MCG, Panax ginseng Meyer) were explored for their diversity and biocontrol activity against ginseng pathogens (Alternaria panax, Botrytis cinerea, Cylindrocarpon destructans, Pythium sp. and Rhizoctonia solani). A total of 1,300 isolates were isolated from three tissues (root, stem and leaf) from MCGs grown in 24 different geographic locations in Korea. In total, 129 different fungal isolates were authenticated by molecular identification based on internal transcribed spacer (ITS) sequences. The fungal endophytes belonged to Ascomycota (81.7%), Basidiomycota (7.08%), Zygomycota (10%) and Unknown (1.15%), with 59 genera. Analysis of diversity indices across sampling sites suggested species abundance as a function of geographical and environmental factors of the locations. Shannon diversity index and richness in the different tissues revealed that root tissues are colonized more than stem and leaf tissues, and also certain fungal endophytes are tissue specific. Assessment of the ethyl acetate extracts from 129 fungal isolates for their biocontrol activity against 5 ginseng pathogens revealed that Trichoderma polysporum produces the antimcriobial metabolite against all the pathogens. This result indicates the promise of its potential usage as a biocontrol agent.

Exposure to Sound Vibrations Lead to Transcriptomic, Proteomic and Hormonal Changes in Arabidopsis.

Sound vibration (SV) is considered as an external mechanical force that modulates plant growth and development like other mechanical stimuli (e.g., wind, rain, touch and vibration). A number of previous and recent studies reported developmental responses in plants tailored against SV of varied frequencies. This strongly suggests the existence of sophisticated molecular mechanisms for SV perception and signal transduction. Despite this there exists a huge gap in our understanding regarding the SV-mediated molecular alterations, which is a prerequisite to gain insight into SV-mediated plant development. Herein, we investigated the global gene expression changes in Arabidopsis thaliana upon treatment with five different single frequencies of SV at constant amplitude for 1 h. As a next step, we also studied the SV-mediated proteomic changes in Arabidopsis. Data suggested that like other stimuli, SV also activated signature cellular events, for example, scavenging of reactive oxygen species (ROS), alteration of primary metabolism, and hormonal signaling. Phytohormonal analysis indicated that SV-mediated responses were, in part, modulated by specific alterations in phytohormone levels; especially salicylic acid (SA). Notably, several touch regulated genes were also up-regulated by SV treatment suggesting a possible molecular crosstalk among the two mechanical stimuli, sound and touch. Overall, these results provide a molecular basis to SV triggered global transcriptomic, proteomic and hormonal changes in plant.

Constitutive over-expression of rice ClpD1 protein enhances tolerance to salt and desiccation stresses in transgenic Arabidopsis plants.

Caseinolytic proteases (Clps) perform the important role of removing protein aggregates from cells, which can otherwise prove to be highly toxic. ClpD system is a two-component protease complex composed of a regulatory ATPase module ClpD and a proteolytic component ClpP. Under desiccation stress condition, rice ClpD1 (OsClpD1) gene encoding for the regulatory subunit, was represented by four variant transcripts differing mainly in the expanse of their N-terminal amino acids. These transcripts were expressed in a differential manner in response to salt, mannitol and polyethylene glycol stresses in rice. Purified OsClpD1.3 protein exhibited intrinsic chaperone activity, shown using citrate synthase as substrate. Arabidopsis (Col-0) plants over-expressing OsClpD1.3 open reading frame downstream to CaMV35S promoter (ClpD1.3 plants) showed higher tolerance to salt and desiccation stresses as compared to wild type plants. ClpD1.3 seedlings also showed enhanced growth during the early stages of seed germination under unstressed, control conditions. The free proline levels and starch breakdown activities were higher in the ClpD1.3 seedlings as compared to the wild type Arabidopsis seedlings. It thus emerges that increasing the potential of ClpD1 chaperoning activity may be of advantage in protection against abiotic stresses.

Plant acoustics: in the search of a sound mechanism for sound signaling in plants.

Being sessile, plants continuously deal with their dynamic and complex surroundings, identifying important cues and reacting with appropriate responses. Consequently, the sensitivity of plants has evolved to perceive a myriad of external stimuli, which ultimately ensures their successful survival. Research over past centuries has established that plants respond to environmental factors such as light, temperature, moisture, and mechanical perturbations (e.g. wind, rain, touch, etc.) by suitably modulating their growth and development. However, sound vibrations (SVs) as a stimulus have only started receiving attention relatively recently. SVs have been shown to increase the yields of several crops and strengthen plant immunity against pathogens. These vibrations can also prime the plants so as to make them more tolerant to impending drought. Plants can recognize the chewing sounds of insect larvae and the buzz of a pollinating bee, and respond accordingly. It is thus plausible that SVs may serve as a long-range stimulus that evokes ecologically relevant signaling mechanisms in plants. Studies have suggested that SVs increase the transcription of certain genes, soluble protein content, and support enhanced growth and development in plants. At the cellular level, SVs can change the secondary structure of plasma membrane proteins, affect microfilament rearrangements, produce Ca(2+) signatures, cause increases in protein kinases, protective enzymes, peroxidases, antioxidant enzymes, amylase, H(+)-ATPase / K(+) channel activities, and enhance levels of polyamines, soluble sugars and auxin. In this paper, we propose a signaling model to account for the molecular episodes that SVs induce within the cell, and in so doing we uncover a number of interesting questions that need to be addressed by future research in plant acoustics.

Characterization of 5'UTR of rice ClpB-C/Hsp100 gene: evidence of its involvement in post-transcriptional regulation.

Rice (Oryza sativa) ClpB-C (OsClpB-C) protein is expressed upon heat stress in vegetative tissues and constitutively in seeds. We produced stably transformed Arabidopsis plants carrying ß-glucuronidase (Gus) reporter gene downstream to 1-kb OsClpB-C promoter (1kbPro plants). In the 1kbPro plants, expression of Gus transcript and protein followed the expression pattern of OsClpB-C gene in rice plants, i.e., heat induced in vegetative tissues and constitutive in seeds. Next, we produced transgenic Arabidopsis plants containing Gus downstream to 862-bp fragment of OsClpB-C promoter [lacking 138 nucleotides from 3' end of the 5'untranslated region (5'UTR); ∆UTR plants). In ∆UTR plants, Gus transcript was expressed in heat-inducible manner, but strikingly, Gus protein levels were negligible after heat treatment. However, Gus protein was expressed in ∆UTR seedlings at levels comparable to 1kbPro seedlings when recovery treatment of 22 °C/2 h was given post heat stress (38 °C/15 min). This suggests that 5'UTR of OsClpB-C gene is involved in its post-transcriptional regulation and is an obligate requirement for protein expression during persistent heat stress. Furthermore, the Gus transcript levels were higher in the polysomal RNA fraction in heat-stressed seedlings of 1kbPro plants as compared to ∆UTR plants, indicating that 5'UTR aids in assembly of ribosomes onto the Gus transcript during heat stress. Unlike the case of seedlings, Gus protein was formed constitutively in ∆UTR seeds at levels comparable to 1kbPro seeds. Hence, the function of 5'UTR of OsClpB-C is dispensable for expression in seeds.

ClpB/Hsp100 proteins and heat stress tolerance in plants.

High-temperature stress can disrupt cellular proteostasis, resulting in the accumulation of insoluble protein aggregates. For survival under stressful conditions, it is important for cells to maintain a pool of native soluble proteins by preventing and/or dissociating these aggregates. Chaperones such as GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE (Hsp70/Hsp40/nucleotide exchange factor) help cells minimize protein aggregation. Protein disaggregation is accomplished by chaperones belonging to the Caseinolytic Protease (Clp) family of proteins. ClpB/Hsp100 proteins are strikingly ubiquitous and are found in bacteria, yeast and multi-cellular plants. The expression of these proteins is regulated by heat stress (HS) and developmental cues. Bacteria and yeast contain one and two forms of ClpB proteins, respectively. Plants possess multiple forms of these proteins that are localized to different cellular compartments (i.e. cytoplasm/nucleus, chloroplast or mitochondria). Overwhelming evidence suggests that ClpB/Hsp100 proteins play decisive roles in cell adaptation to HS. Mutant bacteria and yeast cells lacking active ClpB/Hsp100 proteins are critically sensitive to high-temperature stress. Likewise, Arabidopsis, maize and rice mutants lacking cytoplasmic ClpB proteins are very sensitive to heat. In this study, we present the structural and functional attributes of plant ClpB forms.

Intergenic sequence between Arabidopsis caseinolytic protease B-cytoplasmic/heat shock protein100 and choline kinase genes functions as a heat-inducible bidirectional promoter.

In Arabidopsis (Arabidopsis thaliana), the At1g74310 locus encodes for caseinolytic protease B-cytoplasmic (ClpB-C)/heat shock protein100 protein (AtClpB-C), which is critical for the acquisition of thermotolerance, and At1g74320 encodes for choline kinase (AtCK2) that catalyzes the first reaction in the Kennedy pathway for phosphatidylcholine biosynthesis. Previous work has established that the knockout mutants of these genes display heat-sensitive phenotypes. While analyzing the AtClpB-C promoter and upstream genomic regions in this study, we noted that AtClpB-C and AtCK2 genes are head-to-head oriented on chromosome 1 of the Arabidopsis genome. Expression analysis showed that transcripts of these genes are rapidly induced in response to heat stress treatment. In stably transformed Arabidopsis plants harboring this intergenic sequence between head-to-head oriented green fluorescent protein and ß-glucuronidase reporter genes, both transcripts and proteins of the two reporters were up-regulated upon heat stress. Four heat shock elements were noted in the intergenic region by in silico analysis. In the homozygous transfer DNA insertion mutant Salk_014505, 4,393-bp transfer DNA is inserted at position -517 upstream of ATG of the AtClpB-C gene. As a result, AtCk2 loses proximity to three of the four heat shock elements in the mutant line. Heat-inducible expression of the AtCK2 transcript was completely lost, whereas the expression of AtClpB-C was not affected in the mutant plants. Our results suggest that the 1,329-bp intergenic fragment functions as a heat-inducible bidirectional promoter and the region governing the heat inducibility is possibly shared between the two genes. We propose a model in which AtClpB-C shares its regulatory region with heat-induced choline kinase, which has a possible role in heat signaling.

OsHsfA2c and OsHsfB4b are involved in the transcriptional regulation of cytoplasmic OsClpB (Hsp100) gene in rice (Oryza sativa L.).

ClpB-cytoplasmic (ClpB-cyt)/Hsp100 is an important chaperone protein in rice. Cellular expression of OsClpB-cyt transcript is governed by heat stress, metal stress, and developmental cues. Transgenic rice plants produced with 2 kb OsClpB-cyt promoter driving Gus reporter gene showed heat- and metal-regulated Gus expression in vegetative tissues and constitutive Gus expression in calli, flowering tissues, and embryonal half of seeds. Rice seedlings regenerated with OsClpB-cyt promoter fragment with deletion of its canonical heat shock element sequence (HSE(-273 to -280)) showed not only heat shock inducibility of Gus transcript/protein but also constitutive expression of Gus in vegetative tissues. It thus emerges that the only classical HSE present in OsClpB-cyt promoter is involved in repressing expression of OsClpB-cyt transcript under unstressed control conditions. Yeast one-hybrid assays suggested that OsHsfA2c specifically interacts with OsClpB-cyt promoter. OsHsfA2c also showed binding with OsClpB-cyt and OsHsfB4b showed binding with OsClpB-cyt; notably, interaction of OsHsfB4b was seen for all three OsClpB/Hsp100 protein isoforms (i.e., ClpB-cytoplasmic, ClpB-mitochondrial, and ClpB-chloroplastic). Furthermore, OsHsfB4b showed interaction with OsHsfA2c. This study suggests that OsHsfA2c may play a role as transcriptional activator and that OsHsfB4b is an important part of this heat shock responsive circuitry.