Effects of Elevated Root-Zone CO2 on Root Morphology and Nitrogen Metabolism Revealed by Physiological and Transcriptome Analysis in Oriental Melon Seedling Roots.

Rhizosphere CO2 is vital for crop growth, development, and productivity. However, the mechanisms of plants' responses to root-zone CO2 are unclear. Oriental melons are sensitive to root-zone gas, often encountering high root-zone CO2 during cultivation. We investigated root growth and nitrogen metabolism in oriental melons under T1 (0.5%) and T2 (1.0%) root-zone CO2 concentrations using physiology and comparative transcriptome analysis. T1 and T2 increased root vigor and the nitrogen content in the short term. With increased treatment time and CO2 concentration, root inhibition increased, characterized by decreased root absorption, incomplete root cell structure, accelerated starch accumulation and hydrolysis, and cell aging. We identified 1280 and 1042 differentially expressed genes from T1 and T2, respectively, compared with 0.037% CO2-grown plants. Among them, 683 co-expressed genes are involved in stress resistance and nitrogen metabolism (enhanced phenylpropanoid biosynthesis, hormone signal transduction, glutathione metabolism, and starch and sucrose metabolism). Nitrogen metabolism gene expression, enzyme activity, and nitrogen content analyses showed that short-term elevated root-zone CO2 mainly regulated plant nitrogen metabolism post-transcriptionally, and directly inhibited it transcriptionally in the long term. These findings provided a basis for further investigation of nitrogen regulation by candidate genes in oriental melons under elevated root-zone CO2.

Proteomic discovery of H2O2 response in roots and functional characterization of PutGLP gene from alkaligrass.

MAIN CONCLUSION: Hydrogen peroxide-responsive pathways in roots of alkaligrass analyzed by proteomic studies and PutGLP enhance the plant tolerance to saline-, alkali- and cadmium-induced oxidative stresses. Oxidative stress adaptation is critical for plants in response to various stress environments. The halophyte alkaligrass (Puccinellia tenuiflora) is an outstanding pasture with strong tolerance to salt and alkali stresses. In this study, iTRAQ- and 2DE-based proteomics approaches, as well as qRT-PCR and molecular genetics, were employed to investigate H2O2-responsive mechanisms in alkaligrass roots. The evaluation of membrane integrity and reactive oxygen species (ROS)-scavenging systems, as well as abundance patterns of H2O2-responsive proteins/genes indicated that Ca2+-mediated kinase signaling pathways, ROS homeostasis, osmotic modulation, and transcriptional regulation were pivotal for oxidative adaptation in alkaligrass roots. Overexpressing a P. tenuiflora germin-like protein (PutGLP) gene in Arabidopsis seedlings revealed that the apoplastic PutGLP with activities of oxalate oxidase and superoxide dismutase was predominantly expressed in roots and played important roles in ROS scavenging in response to salinity-, alkali-, and CdCl2-induced oxidative stresses. The results provide insights into the fine-tuned redox-responsive networks in halophyte roots.

Water deficit mechanisms in perennial shrubs Cerasus humilis leaves revealed by physiological and proteomic analyses.

BACKGROUND: Drought (Water deficit, WD) poses a serious threat to extensively economic losses of trees throughout the world. Chinese dwarf cherry (Cerasus humilis) is a good perennial plant for studying the physiological and sophisticated molecular network under WD. The aim of this study is to identify the effect of WD on C. humilis through physiological and global proteomics analysis and improve understanding of the WD resistance of plants. METHODS: Currently, physiological parameters were applied to investigate C. humilis response to WD. Moreover, we used two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in C. humilis leaves subjected to WD (24 d). Furthermore, we also examined the correlation between protein and transcript levels. RESULTS: Several physiological parameters, including relative water content and Pn were reduced by WD. In addition, the malondialdehyde (MDA), relative electrolyte leakage (REL), total soluble sugar, and proline were increased in WD-treated C. humilis. Comparative proteomic analysis revealed 46 protein spots (representing 43 unique proteins) differentially expressed in C. humilis leaves under WD. These proteins were mainly involved in photosynthesis, ROS scavenging, carbohydrate metabolism, transcription, protein synthesis, protein processing, and nitrogen and amino acid metabolisms, respectively. CONCLUSIONS: WD promoted the CO2 assimilation by increase light reaction and Calvin cycle, leading to the reprogramming of carbon metabolism. Moreover, the accumulation of osmolytes (i.e., proline and total soluble sugar) and enhancement of ascorbate-glutathione cycle and glutathione peroxidase/glutathione s-transferase pathway in leaves could minimize oxidative damage of membrane and other molecules under WD. Importantly, the regulation role of carbohydrate metabolisms (e. g. glycolysis, pentose phosphate pathways, and TCA) was enhanced. These findings provide key candidate proteins for genetic improvement of perennial plants metabolism under WD.

Genome-wide identification of CXE and PuCXE15 functions in the catabolism of volatile ester in 'Nanguo' pear fruit.

Volatile esters are the main aromatic components that affect consumer sensory preferences. Aroma is a crucial characteristic of the 'Nanguo' pear (Pyrus ussriensis Maxim). Carboxylesterases (CXEs) are positively correlated with the catabolism of volatile esters in peaches; however, the mechanism of action of CXE family members in 'Nanguo' pear is poorly understood. In this study, 40 PuCXEs were identified in the 'Nanguo' pear and assigned into seven groups. In addition, we found that most PuCXEs were relatively conserved and contained cytoplasmic proteins. This hypothesis was supported by phylogenetic analysis, investigation of conserved domains and gene structures, and prediction of subcellular localization. Based on the content of volatile esters and expression levels of PuCXEs analysis, four PuCXEs, including PuCXE7, PuCXE15, PuCXE20, and PuCXE25, had a significant negative correlation with volatile ester accumulation. Particularly, the correlation of PuCXE15 far exceeded that of the other PuCXEs. The results of the transient expression assay showed that PuCXE15 promoted the degradation of ester in vivo. Subcellular localization experiment revealed that PuCXE15 is located in the plasma membrane and nucleus. These results show that PuCXE15 functions in the catabolism of volatile ester in 'Nanguo' pear fruit, and provides a foundation for enhancing aroma quality by artificial control in pear.

Exogenous melatonin enhances tomato heat resistance by regulating photosynthetic electron flux and maintaining ROS homeostasis.

Heat stress reduces plant growth and reproduction and increases agricultural risks. As a natural compound, melatonin modulates broad aspects of the responses of plants to various biotic and abiotic stresses. However, regulation of the photosynthetic electron transfer, reactive oxygen species (ROS) homeostasis and the redox state of redox-sensitive proteins in the tolerance to heat stress induced by melatonin remain largely unknown. The oxygen evolution complex activity on the electron-donating side of photosystem II (PSII) is inhibited, and the electron transfer process from QA to QB on the electron-accepting side of PSII is inhibited. In this case, heat stress decreased the chlorophyll content, carbon assimilation rate, PSII activity, and the proportion of light absorbed by tomato seedlings during electron transfer. The ROS burst led to the breakdown of the PSII core protein. However, exogenous melatonin increased the net photosynthetic rate by 11.3% compared with heat stress, substantially reducing the restriction of photosynthetic systems induced by heat stress. Additionally, melatonin reduces the oxidative damage to PSII by balancing electron transfer on the donor, reactive center, and acceptor sides. Melatonin was used under heat stress to increase the activity of the antioxidant enzyme and preserve ROS equilibrium. In addition, redox proteomics also showed that melatonin controls the redox levels of proteins involved in photosynthesis, and stress and defense processes, which enhances the expression of oxidative genes. In conclusion, melatonin via controlling the photosynthetic electron transport and antioxidant, melatonin increased tomato heat stress tolerance and aided plant growth.

SlSnRK2.3 interacts with SlSUI1 to modulate high temperature tolerance via Abscisic acid (ABA) controlling stomatal movement in tomato.

Tomato is often exposed to high temperature stress during summer cultivation. Stomatal movement plays important roles in photosynthesis and transpiration which restricts the quality and yield of tomato under environmental stress. To elucidate the mechanism of stomatal movement in high temperature tolerance, SlSnRK2s (sucrose non-fermenting 1-related protein kinases) silenced plants were generated in tomato with CRISPR-Cas 9 gene editing techniques. Through the observation of stomatal parameters, SlSnRK2.3 regulated stomatal closure which was responded to ABA (abscisic acid) and activated signaling pathway of ROS (reactive oxygen species) in high temperature stress. Based on the positive functions of SlSnRK2.3, the cDNA library was generated to investigate interaction proteins of SlSnRK2s. The interaction between SlSnRK2.3 and SlSUI1 (protein translation factor SUI1 homolog) was employed by Yeast two hybrid assay (Y2H), Luciferase (LUC), and Bimolecular fluorescence complementation (BiFC). Finally, the specific interactive sites between SlSnRK2.3 and SlSUI1 were verified by site-directed mutagenesis. The consistent mechanism of SlSnRK2.3 and SlSUI1 in stomatal movement, indicating that SlSUI1 interacted with SlSnRK2.3 through ABA-dependent signaling pathway in high temperature stress. Our results provided evidence for improving the photosynthetic capacity of tomato under high temperature stress, and support the breeding and genetic engineering of tomato over summer facility cultivation.

Integrated metabolome and transcriptome analysis of the regulatory network of volatile ester formation during fruit ripening in pear.

'Nanguo' pear (Pyrus ussuriensis Maxim.) is a typical climacteric fruit with an attractive aroma after postharvest ripening. Esters are the key volatile compounds determining the typical aroma formation. However, the mechanism of aroma-related ester formation remains largely unknown. In this study, we performed transcriptome and metabolome analyses to reveal the changes of aroma-related compounds during pear ripening in the optimal taste period (OTP). During the pear ripening process, typical fatty acid-derived volatile organic compounds (VOCs) are transformed from aldehydes, alcohols, and ketones to esters, where ethyl hexanoate, hexyl acetate, and ethyl butanoate are the dominant esters in the OTP. Rich aroma-related esters in the OTP are associated with the accumulation of important precursors of aroma volatiles, including linoleic acid, α-linolenic acid, γ-linolenic acid, and oleic acid. Genes encoding key biosynthetic enzymes are associated with the altered levels of aroma-related esters. The candidate genes associated with the high levels of aroma-related esters in 'Nanguo' pears are PuFAD2, PuLOX2, PuLOX5, and PuAAT. Additionally, transcription factor (TF) genes such as PuWRKY24, PuIAA29, and PuTINY may play crucial roles in aroma formation during fruit ripening. Hence, we summarized the TFs that regulate VOC metabolism in different fruit species. The results provided a foundation for further research on aroma-related esters in 'Nanguo' pears and could help to elucidate the mechanisms regulating fruit quality improvement.

Differential responses of 23 maize cultivar seedlings to an arbuscular mycorrhizal fungus when grown in a metal-polluted soil.

Modern breeding efforts have been accelerating crop improvement and yielding numerous cultivars with distinct genetic traits; however, interactions between different cultivars and their root-associated arbuscular mycorrhizal fungi (AMF) are not clear. Herein, we selected the 22 most common commercial maize (Zea mays) varieties in China and an inbred line (B73) to study the differential responses of these 23 cultivars to mycorrhizal inoculation when grown in an arable soil polluted by multiple metals (Pb, Zn, and Cd). We found that the different cultivars exhibited significant variations in plant metal accumulation, ranging from strong metal exclusion (ZYY9) to strong metal accumulation (B73). Mycorrhizal colonization substantially altered metal uptake and repartitioning, while bioaugmenting the inherent characteristics of metal accumulation; for example, the AMF enhanced leaf accumulation of the metal-accumulator B73, and markedly reduced the root uptake of the metal-excluder ZYY9. However, such AMF-induced alterations were also substantially dependent on plant organs (roots and shoots) and metal species. We found that the extent of the AMF-induced leaf alterations was substantially greater than that of the root alterations. Similarly, the number of instances where the AMF significantly altered the Zn and Cd accumulation was far higher than the number of instances where Pb accumulation was significantly altered by AMF. In addition, the presence of AMF appeared to trigger the maize antioxidant systems, which may have alleviated the toxicity of excessive Cd, increased the leaf chlorophyll content, augmented the net photosynthetic rate, and promoted the growth of 17.39% of the maize cultivars. Our results suggest that a future crop breeding challenge is to produce cultivars for safe production or phytoremediation, thereby optimizing the combinations of crop cultivars and their root-associated AMF in slightly to moderately metal-polluted arable soils.

Cyclic electron flow modulate the linear electron flow and reactive oxygen species in tomato leaves under high temperature.

The cyclic electron flow (CEF) around photosystem I (PSI) plays a crucial role in photosynthesis and also functions in plant tolerance of abiotic environmental stress. However, the role of PGR5/PGRL1- and NDH-dependent CEF in tomato under hightemperature (HT) is poorly understood. Here, we assessed the photoprotective effect of these pathways in tomato leaves under HT by using antimycin A (AA) and rotenone (R), which are chemical inhibitors of PGR5/PGRL1- and NDH-dependent CEF, respectively. The results showed that AA treatment caused significantly greater inhibition of CEF under HT compared to R treatment. Moreover, AA treatment caused a greater decrease in maximal photochemistry efficiency (Fv/Fm) and increased damage to the donor and acceptor side of photosystem II (PSII); however, the limitation of the acceptor side in PSI [Y(NA)] was significantly increased. In addition, thylakoid membrane integrity was compromised and reactive oxygen species, proton gradient (ΔpH), antioxidant enzyme activity, and the expression of photosystem core subunit genes were significantly decreased under AA treatment. These findings indicate that PGR5/PGRL1-dependent CEF protects PSII and PSI from photooxidative damage through the formation of ΔpH while maintaining thylakoid membrane integrity and normal gene expression levels of core photosystem components. This study demonstrates that PGR5/PGRL1-dependent CEF plays a major role in HT response in tomato.

Quantitative proteomics reveals a role of JAZ7 in plant defense response to Pseudomonas syringae DC3000.

Jasmonate ZIM-domain (JAZ) proteins are key transcriptional repressors regulating various biological processes. Although many studies have studied JAZ proteins by genetic and biochemical analyses, little is known about JAZ7-associated global protein networks and how JAZ7 contributes to bacterial pathogen defense. In this study, we aim to fill this knowledge gap by conducting unbiased large-scale quantitative proteomics using tandem mass tags (TMT). We compared the proteomes of a JAZ7 knock-out line, a JAZ7 overexpression line, as well as the wild type Arabidopsis plants in the presence and absence of Pseudomonas syringae DC3000 infection. Both pairwise comparison and multi-factor analysis of variance reveal that differential proteins are enriched in biological processes such as primary and secondary metabolism, redox regulation, and response to stress. The differential regulation in these pathways may account for the alterations in plant size, redox homeostasis and accumulation of glucosinolates. In addition, possible interplay between genotype and environment is suggested as the abundance of seven proteins is influenced by the interaction of the two factors. Collectively, we demonstrate a role of JAZ7 in pathogen defense and provide a list of proteins that are uniquely responsive to genetic disruption, pathogen infection, or the interaction between genotypes and environmental factors. SIGNIFICANCE: We report proteomic changes as a result of genetic perturbation of JAZ7, and the contribution of JAZ7 in plant immunity. Specifically, the similarity between the proteomes of a JAZ7 knockout mutant and the wild type plants confirmed the functional redundancy of JAZs. In contrast, JAZ7 overexpression plants were much different, and proteomic analysis of the JAZ7 overexpression plants under Pst DC3000 infection revealed that JAZ7 may regulate plant immunity via ROS modulation, energy balance and glucosinolate biosynthesis. Multiple variate analysis for this two-factor proteomics experiment suggests that protein abundance is determined by genotype, environment and the interaction between them.

Rootstock Alleviates Salt Stress in Grafted Mulberry Seedlings: Physiological and PSII Function Responses.

This study investigated the effect of NaCl stress on Na+ and K+ absorption and transport by roots, nitrogen and phosphorus content in leaves, PSII photochemical activity and reactive oxygen species (ROS) in leaves of mulberry own-root seedlings and grafted seedlings. To determine the response, own-root seedlings of a high yielding mulberry cultivar, Tieba mulberry (Morus alba L.), and the grafted seedlings, obtained by using Qinglong mulberry with high salt tolerance as rootstock and Tieba mulberry as scion, were used. The Na+ content in roots and leaves of grafted seedlings was significantly lower than that in own-root seedlings under salt stress; while K+ content in roots and leaves of grafted seedlings was significantly higher than that in own-root seedlings. The root activity in grafted seedlings was significantly higher than that in own-root seedlings, as well as the content of nitrogen, phosphorous and water. PSII photochemical activity in leaves of grafted seedlings was less significantly affected by salt stress compared to own-root seedlings. The electron transport at the acceptor side of PSII from Q A to Q B was less affected by salt stress, which resulted in a significantly lower ROS content in leaves of grafted seedlings than that of own-root seedlings. Therefore, grafting high-yielding and good-quality Tieba mulberry with salt tolerant Qinglong mulberry as rootstock showed a relatively high salt tolerance. This may be because (1) the root system of rootstock presented high Na+ resistance and has selective absorption capacity for Na+ and K+ (2) the root system of rootstock prevented excess Na+ from being transported to aerial parts in order to reduce adverse effects of Na+ (3) the root system of rootstock had enhanced root activity under salt stress, which accelerated water and nutrient absorption (4) the leaves of grafted seedlings had higher PSII photochemical activity and electron transport rate compared with those of own-root seedlings under salt stress, which effectively reduced ROS burst mediated by photosynthesis and reduced oxidative damage.

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells.

Climate change as a result of increasing atmospheric CO2 affects plant growth and productivity. CO2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO2) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO2 responses.

Metabolomic Responses of Arabidopsis Suspension Cells to Bicarbonate under Light and Dark Conditions.

Global CO2 level presently recorded at 400 ppm is expected to reach 550 ppm in 2050, an increment likely to impact plant growth and productivity. Using targeted LC-MS and GC-MS platforms we quantified 229 and 29 metabolites, respectively in a time-course study to reveal short-term responses to different concentrations (1, 3, and 10 mM) of bicarbonate (HCO3-) under light and dark conditions. Results indicate that HCO3- treatment responsive metabolomic changes depend on the HCO3- concentration, time of treatment, and light/dark. Interestingly, 3 mM HCO3- concentration treatment induced more significantly changed metabolites than either lower or higher concentrations used. Flavonoid biosynthesis and glutathione metabolism were common to both light and dark-mediated responses in addition to showing concentration-dependent changes. Our metabolomics results provide insights into short-term plant cellular responses to elevated HCO3- concentrations as a result of ambient increases in CO2 under light and dark.

Chilling-responsive mechanisms in halophyte Puccinellia tenuiflora seedlings revealed from proteomics analysis.

Alkali grass (Puccinellia tenuiflora), a monocotyledonous perennial halophyte species, is a good pasture with great nutritional value for livestocks. It can thrive under low temperature in the saline-alkali soil of Songnen plain in northeastern China. In the present study, the chilling-responsive mechanism in P. tenuiflora leaves was investigated using physiological and proteomic approaches. After treatment of 10°C for 10 and 20days, photosynthesis, biomass, contents of osmolytes and antioxidants, and activities of reactive oxygen species scavenging enzymes were analyzed in leaves of 20-day-old seedlings. Besides, 89 chilling-responsive proteins were revealed from proteomic analysis. All the results highlighted that the growth of seedlings was inhibited due to chilling-decreased enzymes in photosynthesis, carbohydrate metabolism, and energy supplying. The accumulation of osmolytes (i.e., proline, soluble sugar, and glycine betaine) and enhancement of ascorbate-glutathione cycle and glutathione peroxidase/glutathione S-transferase pathway in leaves could minimize oxidative damage of membrane and other molecules under the chilling conditions. In addition, protein synthesis and turnover in cytoplasm and chloroplast were altered to cope with the chilling stress. This study provides valuable information for understanding the chilling-responsive and cross-tolerant mechanisms in monocotyledonous halophyte plant species.

Na2CO3-responsive mechanisms in halophyte Puccinellia tenuiflora roots revealed by physiological and proteomic analyses.

Soil alkalization severely affects crop growth and agricultural productivity. Alkali salts impose ionic, osmotic, and high pH stresses on plants. The alkali tolerance molecular mechanism in roots from halophyte Puccinellia tenuiflora is still unclear. Here, the changes associated with Na2CO3 tolerance in P. tenuiflora roots were assessed using physiological and iTRAQ-based quantitative proteomic analyses. We set up the first protein dataset in P. tenuiflora roots containing 2,671 non-redundant proteins. Our results showed that Na2CO3 slightly inhibited root growth, caused ROS accumulation, cell membrane damage, and ion imbalance, as well as reduction of transport and protein synthesis/turnover. The Na2CO3-responsive patterns of 72 proteins highlighted specific signaling and metabolic pathways in roots. Ca(2+) signaling was activated to transmit alkali stress signals as inferred by the accumulation of calcium-binding proteins. Additionally, the activities of peroxidase and glutathione peroxidase, and the peroxiredoxin abundance were increased for ROS scavenging. Furthermore, ion toxicity was relieved through Na(+) influx restriction and compartmentalization, and osmotic homeostasis reestablishment due to glycine betaine accumulation. Importantly, two transcription factors were increased for regulating specific alkali-responsive gene expression. Carbohydrate metabolism-related enzymes were increased for providing energy and carbon skeletons for cellular metabolism. All these provide new insights into alkali-tolerant mechanisms in roots.

[Heat-responsive mechanisms in plants revealed by proteomic analysis: A review].

Heat stress is a major abiotic stress that limits plant growth and productivity. In recent years, proteomic investigations provide more information for understanding the sophisticated heat-responsive molecular mechanism in plants at systematic biological level. The heat-responsive proteomic patterns in several plants, i. e., model plants (Arabidopsis thaliana), staple food crops (soybean, rice and wheat), heat-tolerant plants (Agrostis stolonifera, Portulaca oleracea, and Carissa spinarum), grapevine, Populus euphratica, Medicago sativa, and Pinellia ternate, were reported. A total of 838 heat-responsive proteins have been identified in these studies. Among them, 534 proteins were induced and the expression of 304 proteins was reduced in plants under heat stress. In this paper, the diverse protein patterns in plants under various heat stress conditions (30-45 °C for 0-10 d) were analyzed integratively. This provided new evidences and clues for further interpreting the signaling and metabolic pathways, e.g., signaling, stress and defense, carbohydrate and energy metabolism, photosynthesis, transcription, protein synthesis and fate, membrane and transport, in heat-responsive networks, and laid a foundation for a holistic understanding of the molecular regulatory mechanism in plants in response to heat stress.