Genome-Wide Identification and Expression Patterns of the C2H2-Zinc Finger Gene Family Related to Stress Responses and Catechins Accumulation in Camellia sinensis [L.] O. Kuntze

The C2H2-zinc finger protein (C2H2-ZFP) is essential for the regulation of plant development and widely responsive to diverse stresses including drought, cold and salt stress, further affecting the late flavonoid accumulation in higher plants. Tea is known as a popular beverage worldwide and its quality is greatly dependent on the physiological status and growing environment of the tea plant. To date, the understanding of C2H2-ZFP gene family in Camellia sinensis [L.] O. Kuntze is not yet available. In the present study, 134 CsC2H2-ZFP genes were identified and randomly distributed on 15 chromosomes. The CsC2H2-ZFP gene family was classified into four clades and gene structures and motif compositions of CsC2H2-ZFPs were similar within the same clade. Segmental duplication and negative selection were the main forces driving the expansion of the CsC2H2-ZFP gene family. Expression patterns suggested that CsC2H2-ZFPs were responsive to different stresses including drought, salt, cold and methyl jasmonate (MeJA) treatment. Specially, several C2H2-ZFPs showed a significant correlation with the catechins content and responded to the MeJA treatment, which might contribute to the tea quality and specialized astringent taste. This study will lay the foundations for further research of C2H2-type zinc finger proteins on the stress responses and quality-related metabolites accumulation in C. sinensis.     

Genome-Wide Identification of Strawberry C2H2-ZFP C1-2i Subclass and the Potential Function of FaZAT10 in Abiotic Stress

C2H2-type zinc finger proteins (C2H2-ZFPs) play a key role in various plant biological processes and responses to environmental stresses. In Arabidopsis thaliana, C2H2-ZFP members with two zinc finger domains have been well-characterized in response to abiotic stresses. To date, the functions of these genes in strawberries are still uncharacterized. Here, 126 C2H2-ZFPs in cultivated strawberry were firstly identified using the recently sequenced Fragaria × ananassa genome. Among these C2H2-ZFPs, 46 members containing two zinc finger domains in cultivated strawberry were further identified as the C1-2i subclass. These genes were unevenly distributed on 21 chromosomes and classified into five groups according to the phylogenetic relationship, with similar physicochemical properties and motif compositions in the same group. Analyses of conserved domains and gene structures indicated the evolutionary conservation of the C1-2i subclass. A Ka/Ks analysis indicated that the C1-2i members were subjected to purifying selection during evolution. Furthermore, FaZAT10, a typical C2H2-ZFP, was isolated. FaZAT10 was expressed the highest in roots, and it was induced by drought, salt, low-temperature, ABA, and MeJA treatments. It was localized in the nucleus and showed no transactivation activity in yeast cells. Overall, these results provide useful information for enriching the analysis of the ZFPs gene family in strawberry, and they provide support for revealing the mechanism of FaZAT10 in the regulatory network of abiotic stress.     

Enhancing Sorgoleone Levels in Grain Sorghum Root Exudates

Sorgoleone, found in the root exudates of sorghum [(Sorghum bicolor (L.) Moench], has been a subject of continued research. Sorgoleone production in grain sorghum roots was investigated under different growth conditions. Methanol was the most effective solvent for extracting sorgoleone from grain sorghum roots. Sorgoleone production is high in young developing plants. The maximum concentration (μg mg^?1 root dry weight) was produced in 5-d-old seedlings; beyond this age, production declined. However, considering both root weight and sorgoleone content per seedling, 10-d-old seedlings had the highest total amounts (μg). Compared with the control, sorgoleone content increased 6.1, 8.6, and 14.2 times when sorghum seeds were treated with auxins, Hoagland solution, and a combination of auxins and Hoagland solution, respectively. Among the innate immunity response elicitors, cellulose (an elicitor of plant origin) stimulated higher sorgoleone production than the others, and it produced 6.2 times more sorgoleone than the control. Combined treatment of sorghum seeds with half strength Hoagland solution and 5 μg ml^?1 of IBA significantly increased both root growth and sorgoleone content in sorghum seedlings.     

Identification and Analysis of Stress-Associated Proteins (SAPs) Protein Family and Drought Tolerance of ZmSAP8 in Transgenic Arabidopsis

Stress-associated proteins (SAPs), a class of A20/AN1 zinc finger proteins, play vital roles in plant stress response. However, investigation of SAPs in maize has been very limited. Herein, to better trace the evolutionary history of SAPs in maize and plants, 415 SAPs were identified in 33 plant species and four species of other kingdoms. Moreover, gene duplication mode exploration showed whole genome duplication contributed largely to SAP gene expansion in angiosperms. Phylogeny reconstruction was performed with all identified SAPs by the maximum likelihood (ML) method and the SAPs were divided into five clades. SAPs within the same clades showed conserved domain composition. Focusing on maize, nine ZmSAPs were identified. Further promoter cis-elements and stress-induced expression pattern analysis of ZmSAPs indicated that ZmSAP8 was a promising candidate in response to drought stress, which was the only AN1-AN1-C2H2-C2H2 type SAP in maize and belonged to clade I. Additionally, ZmSAP8 was located in the nucleus and had no transactivation activity in yeast. Overexpressing ZmSAP8 enhanced the tolerance to drought stress in Arabidopsis thaliana, with higher seed germination and longer root length. Our results should benefit the further functional characterization of ZmSAPs.     

Identification of Unique Aldehyde Dimers in Sorghum Wax Recovered after Fermentation in a Commercial Fuel Ethanol Plant

Sorghum wax can be extracted from the surface of sorghum (Sorghum bicolor) kernels. It is composed mostly of a mixture of unsaturated C₂₈ and C₃₀ alkanes, fatty acids, fatty alcohols, and fatty aldehydes. Like carnauba wax, sorghum wax is a hard wax with a high melting point and it has potential edible and industrial applications. The yield of sorghum wax from the surface of sorghum kernels is 0.2–0.5 g of wax per 100 g of kernels. Sorghum wax can also be recovered from the “distillers oil” which is obtained after fermentation of sorghum (milo) or sorghum/corn blends in dry grind fuel ethanol plants. This distillers sorghum wax can potentially be obtained in yields of up to 10% by chilling the distillers oil to precipitate the wax and then recovering it via centrifugation or filtration. Like sorghum kernel wax, distillers sorghum wax is mainly composed of C₂₈ and C₃₀ alkanes, alcohols, and aldehydes in the molecular weight (MW) range of 350–450. However, we found that 7–49% w/w of distillers sorghum wax is composed of larger wax components with MW of 799–912. Analysis via high-resolution atmospheric pressure chemical ionization mass spectrometry (APCI) and gas chromatography with electron ionization mass spectrometry (GC/MS-EI) resulted in exact mass data and fragmentation patterns that suggested that these high MW compounds are monounsaturated fatty aldehyde dimers, likely formed by aldol condensation. Further confirmation supporting the GC/MS data for the aldol reaction was obtained by comparison with similar aldol products.     

A C2H2-Type Zinc-Finger Protein from Millettia pinnata,MpZFP1, Enhances Salt Tolerance in Transgenic Arabidopsis

C2H2 zinc finger proteins (ZFPs) play important roles in plant development and response to abiotic stresses, and have been studied extensively. However, there are few studies on ZFPs in mangroves and mangrove associates, which represent a unique plant community with robust stress tolerance. MpZFP1, which is highly induced by salt stress in the mangrove associate Millettia pinnata, was cloned and functionally characterized in this study. MpZFP1 protein contains two zinc finger domains with conserved QALGGH motifs and targets to the nucleus. The heterologous expression of MpZFP1 in Arabidopsis increased the seeds’ germination rate, seedling survival rate, and biomass accumulation under salt stress. The transgenic plants also increased the expression of stress-responsive genes, including RD22 and RD29A, and reduced the accumulation of reactive oxygen species (ROS). These results indicate that MpZFP1 is a positive regulator of plant responses to salt stress due to its activation of gene expression and efficient scavenging of ROS.     

Revealing Differential Expression of Phytohormones in Sorghum in Response to Aphid Attack Using the Metabolomics Approach

Sorghum (Sorghum bicolor) is an important multipurpose crop grown worldwide, but like many other crops, it is often threatened by insect pests. Sugarcane aphid (SCA, Melanaphis sacchari Zehntner), for example, is one of the most severe pests in sorghum, which causes plant damage and yield loss. The main objective of this study was to assess the effect of phytohormones on host plant resistance to aphid attack. Two sorghum genotypes, BTx623 (susceptible) and Tx2783 (resistant), were selected for a comparative analysis of differential expression of a group of phytohormones in response to aphid infestation. The quantification of phytohormones through LC-MS demonstrated higher levels of jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), and auxins in the resistant genotype infested with SCA. The PCA plot supports the strong differential responses between resistant and susceptible genotypes, indicating a positive correlation between JA and ABA and a negative correlation between SA and auxins. Similarly, RT-PCR results of the phytohormones-related marker genes showed higher expression in the resistant genotype compared to the susceptible one. Furthermore, to corroborate the role of phytohormones in plant defense, the susceptible genotype was treated with SA, JA, and ABA. The exogenous application of SA and JA + ABA significantly reduced plant mortality, aphid number, and damage in the susceptible genotype, suggesting a strong correlation between phytohormones and plant survival. Our findings indicate that phytohormones play positive roles in plant defense against aphids and provide new insights into the molecular mechanisms operating in plants for self-protection. These findings could also stimulate further research into the mystery about the regulation of phytohormone production during plant interaction with aphids.     

Proteomic Analysis Revealed Different Molecular Mechanisms of Response to PEG Stress in Drought-Sensitive and Drought-Resistant Sorghums

Drought is the major limiting factor that directly or indirectly inhibits the growth and reduces the productivity of sorghum (Sorghum bicolor (L.) Moench). As the main vegetative organ of sorghum, the response mechanism of the leaf to drought stress at the proteomic level has not been clarified. In the present study, nano-scale liquid chromatography mass spectrometry (nano-LC-MS/MS) technology was used to compare the changes in the protein expression profile of the leaves of drought-sensitive (S4 and S4-1) and drought-resistant (T33 and T14) sorghum varieties at the seedling stage under 25% PEG-6000 treatment for 24 h. A total of 3927 proteins were accurately quantitated and 46, 36, 35, and 102 differentially abundant proteins (DAPs) were obtained in the S4, S4-1, T14, and T33 varieties, respectively. Four proteins were randomly selected for parallel reaction monitoring (PRM) assays, and the results verified the reliability of the mass spectrometry (MS) results. The response mechanism of the drought-sensitive sorghum leaves to drought was attributed to the upregulation of proteins involved in the tyrosine metabolism pathway with defense functions. Drought-resistant sorghum leaves respond to drought by promoting the TCA cycle, enhancing sphingolipid biosynthesis, interfering with triterpenoid metabolite synthesis, and influencing aminoacyl-tRNA biosynthesis. The 17 screened important candidate proteins related to drought stress were verified by quantitative real-time PCR (qRT-PCR), the results of which were consistent with the results of the proteomic analysis. This study lays the foundation for revealing the drought-resistance mechanism of sorghum at the protein level. These findings will help us cultivate and improve new drought-resistant sorghum varieties.     

Effects of molasses grass,Melinis minutiflora volatiles on the foraging behavior of the cereal stemborer parasitoid,Cotesia sesamiae

Olfactory responses of the cereal stemborer parasitoid Cotesia sesamiae to volatiles emitted by gramineous host and nonhost plants of the stemborers were studied in a Y-tube olfactometer. The host plants were maize (Zea mays) and sorghum ( Sorghum bicolor), while the nonhost plant was molasses grass (Melinis minutiflora). In single-choice tests, females of C. sesamiae chose volatiles from infested and uninfested host plants and molasses grass over volatiles from the control (soil). In dual-choice tests, the wasp preferred volatiles from infested host plants to those from uninfested host plants. There was no discrimination between molasses grass volatiles and those of uninfested maize, uninfested sorghum, or infested maize. The wasp preferred sorghum volatiles over maize. Combining uninfested maize or sorghum with molasses grass did not make volatiles from the combination more attractive as compared to only uninfested host plants. Infested maize alone was as attractive as when combined with molasses grass. Infested sorghum was preferred over its combination with molasses grass. Local growth conditions of the molasses grasses influenced attractiveness to the parasitoids. Volatiles from Thika molasses grass were attractive, while those from Mbita molasses grass were not. Growing the Thika molasses grass in Mbita rendered it unattractive and vice versa with the Mbita molasses grass. This is a case of the same genotype expressing different phenotypes due to environmental factors.     

Identification of Small‐Molecule PHD2 Zinc Finger Inhibitors that Activate Hypoxia Inducible Factor

The prolyl hydroxylase domain (PHD) protein:hypoxia inducible factor (HIF) pathway is the main pathway by which changes in oxygen concentration are transduced to changes in gene expression. In mammals, there are three PHD paralogues, and PHD2 has emerged as a particularly critical one for regulating HIF target genes such as erythropoietin (EPO), which controls red cell mass and hematocrit. PHD2 is distinctive among the three PHDs in that it contains an N‐terminal MYND‐type zinc finger. We have proposed that this zinc finger binds a Pro‐Xaa‐Leu‐Glu (PXLE) motif found in proteins of the HSP90 pathway to facilitate HIF‐α hydroxylation. Targeting this motif could provide a means of specifically inhibiting this PHD isoform. Here, we screened a library of chemical compounds for their capacity to inhibit the zinc finger of PHD2. We identified compounds that, in vitro, can inhibit PHD2 binding to a PXLE‐containing peptide and induce activation of HIF. Injection of one of these compounds into mice induces an increase in hematocrit. This study offers proof of principle that inhibition of the zinc finger of PHD2 can provide a means of selectively targeting PHD2 to activate the HIF pathway.