Plant flotillins, a subgroup of the SPFH domain protein superfamily, consist of three proteins, AtFLOT1, AtFLOT2, and AtFLOT3 in Arabidopsis thaliana. The exact functions of flotillins in plant cell has not been established yet. In this study we focused on the role of flotillins in response to both abiotic and biotic stresses and on the response to phytohormones abscisic acid and 1-naphthalene acetic acid (NAA) in A. thaliana. We observed transcriptomic changes of AtFLOT genes in response to high salinity and cold, treatment with 22-amino acid peptide from N-terminal part of flagellin (flg22), and after infection with Botrytis cinerea. Transcription of AtFLOT2 increased up to 60 times after flg22 treatment. Also, treatment with B. cinerea increased transcription of AtFLOT1 10 times and of AtFLOT3 14 times. Furthermore, we used T-DNA knock-out single mutants for all three A. thaliana flotillins and we measured root growth in response to high salinity, cold, phosphate starvation, nitrogen starvation, and abscisic acid and NAA treatments. Subsequently, we measured the reactive oxygen species production and callose accumulation after the treatment with flg22. Next, we performed resistance assays to Pseudomonas syringae pv. tomato DC3000 and B. cinerea. In contrast to transcriptomic changes, knocking-out of only single FLOT gene did not lead to significant changes in response to all tested stresses.

Cell walls play an important role in the structure and morphology of plants as well as in responses to various biotic and abiotic stresses. Although the comprehensive analysis of genes involved in cellulose synthase has been performed in model plants, such as Arabidopsis thaliana and rice, information regarding cellulose synthase-like (Csl) genes in maize is limited. In this study, a total of 56 members of Csl gene family were identified in maize genome and classified into six subfamilies. Analysis of gene structure and conserved motif indicated functional similarities among the ZmCsl proteins within the same subfamily. Additionally, the 56 ZmCsl genes were dispersed on 10 chromosomes. The expression patterns of ZmCsl genes in different tissues using the transcriptome data revealed that most of ZmCsl genes had a relatively high expression in root and tassel tissues. Moreover, the expression profiles of ZmCsl genes under drought and re-watering indicated that the expression of ZmCsl genes were mainly responsive to early stage of drought stress. The protein-protein interaction network of ZmCsl proposed some potentially interacting proteins. The data presented a comprehensive survey of Csl gene family in maize. The detailed description of maize Csl genes will be beneficial to understand their structural, functional, and evolutionary features and provide an important foundation for studying the roles of ZmCsl genes in response to biotic and abiotic stresses.

Plants must precisely regulate their signalling pathways to respond to environmental changes promptly. Sucrose non fermenting1 (SNF1)-related protein kinases (SnRK) 2 are essential kinases in abiotic stress responses, including responses to abscisic acid. Although homologs of SnRKs in yeast require a γ-subunit for full activation, it has been unclear whether SnRK2s in higher plants are affected by γ-subunits. In this report, we aimed to show the effect of Arabidopsis KIN γ-subunit 1 (KING1), which is a potential γ-subunit, on the activity of SnRK2. A recombinant KING1 bound to SnRK2.6 and functionally inhibited its activity in vitro. On the other hand, KING1 facilitated the activity of SnRK2.2. Structural models suggest that significant structural changes occurred as a result of KING1 binding to the C-terminal tail of SnRK2s. Since KING1 inhibited the kinase activity of a chimeric protein consisting of the N-terminal domain of SnRK2.6 and the C-terminal domain of SnRK2.2, regulation by KING1 was determined by the N-terminal domain of SnRK2s. Together, these results show that KING1 can mediate activity of SnRK2s in vitro.

Plant hormones play important roles in seed dormancy and dormancy breaking. We measured the hormone content in rice (Oryza sativa L. cv. Nona Bokra) seeds at different stages and with or without imbibition treatment. We identified 1 265 differentially expressed genes (DEGs) between dormant and dormancy-broken seeds using RNA-seq analysis: 1 015 genes were significantly up-regulated, while 250 genes were significantly down-regulated. Sixteen DEGs were selected as related to seed dormancy, and their expressions were validated using quantitative PCR. Three DEGs were in the same position as two reported dormancy QTLs, suggesting that they may be candidate genes that control the dormancy of rice seeds. Our study provides an important basis for cloning genes in dormant rice seeds and provides theoretical support for the study of the dormancy mechanism.

Reverse genetics approaches in plants rely on post-transcriptional gene silencing to study the function of genes. In particular, virus-induced gene silencing (VIGS) has been successfully applied to identify gene function in some crops. To date, it is unclear whether phenylalanine ammonia-lyase (PAL) is involved in low temperature tolerance in the pepper. Here, we used an agroinfiltration protocol with tobacco rattle virus (TRV) constructs containing partial sequences from CaPAL for VIGS to test its role in anthocyanin biosynthesis and response to low temperature in the pepper (Capsicum annuum). We found that accumulation of anthocyanins in the leaves of pepper plants transformed with the TRV2:CaPAL vector was significantly reduced compared with peppers transformed with the empty TRV2 vector (TRV2:00). A significant reduction in expression of genes related to anthocyanins synthesis was also detected in peppers transformed with TRV2:CaPAL. When silenced pepper plants were exposed to a low temperature, we found decreased antioxidant system, PAL activity, and photosynthesis in plants transformed with TRV2:CaPAL compared with peppers transformed with TRV2:00. Low transcriptions of cold stress-response genes demonstrated that pepper tolerance to low temperature decreased. Future studies focused on the interaction between CaPAL and other abiotic and biotic stressors will shed further light into the role of CaPAL in stress response.

Proline rich proteins (PRPs), earlier famous as animal salivary proteins, have now been proven as indispensable plant proteins. They are highly rich in proline amino acid residues at the N-terminus whereas a characteristic eight cysteine motif is located at the C-terminus. The PRPs support a number of developmental processes from germination to plant death. Under normal environmental conditions, PRP genes express customarily in different plant parts depending on the specific function to be carried out. During abiotic stresses, PRP genes exhibit an uneven pattern of transcriptional regulation depending on the time and intensity of stress. Transgenic plants overexpressing PRP genes show an enhanced tolerance to abiotic stresses. This review focuses on contemporary functions of PRPs during stresses and proposes that PRPs are involved in the regulation of free cellular proline content during stress in a well synchronized manner.

Osmanthus fragrans Lour., one of the top 10 most popular flowers in China, is known for both its beauty and fragrance. It is rich in flavonoids, a class of secondary metabolites with significant neuroprotective, free-radical scavenging, and anti-oxidant activity. To understand the mechanisms regulating flavonoid biosynthesis, we conducted transcriptome sequencing O. fragrans flowers to analyze gene expressions during the full flowering stage. The RNA was isolated separately from petals of cvs. Yingui and Dangui, which were treated or not with jasmonic acid, salicylic acid, or abscisic acid. A total of 142 029 unigenes were denovo assembled, and 50 918 unigenes were annotated. The differentially expressed genes were identified, annotated, and classified. The results of transcriptome sequencing and real-time PCR revealed higher expressions of phenylalanine ammonia-lyase (PAL), PAL1, chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), flavonol synthase (FLS), and lower expressions of dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS) in 'Yingui' than in 'Dangui'. Such an expression pattern facilitated the higher accumulation of flavonoids in 'Yingui'. Several genes of the flavonoid biosynthesis pathway were upregulated by jasmonic acid and salicylic acid in both the cultivars leading to flavonoid accumulation in their petals. In the v-myb avian myeloblastosis viral oncogene homolog 1(MYB1)-overexpressing petals, the expressions of PAL, PAL1, CHI, and FLS increased. The results suggest that MYB1 may participate in the flavonoid biosynthesis pathway and regulate the expression of some upstream genes in O. fragrans.

The rice OsMyb4 gene, which encodes a Myb transcription factor (TF), improves the stress tolerance/resistance when expressed in both monocotyledonous and dicotyledonous transgenic plants. In this study, a phylogenetic analysis showed the existence of putative OsMyb4 homologues in monocot and dicot species. In particular, the analysis revealed that OsMyb4 belongs to a small rice gene subfamily conserved among monocots. The expression analyses of the OsMyb4-like genes in rice, wheat, and Arabidopsis indicated that these genes are involved in the response to dehydration, cold, and wounding. Moreover, the in silico analysis of the 5' upstream regions of the Osmyb4-like genes highlighted that the positions of some cis-elements involved in the stress response were conserved among the putative promoters, especially between OsMyb4 and its putative paralog Os02g41510. Finally, our transient expression assays in tobacco protoplasts demonstrated that OsMyb4 is able to repress the activity of both its own promoter and the Os02g41510 promoter by acting on the same binding site. A compensatory mechanism of auto-regulation is consistent with the well-known complexity of the OsMyb4-activated pathway, and this mechanism could regulate the transcription of other genes belonging to the family.

Virus-induced gene silencing (VIGS) is a particularly useful tool for functional genomics. In the present study, we attempted to utilize this technology to infer the function of genes from Viola philippica using a tobacco rattle virus (TRV) construct. Firstly, the phytoene desaturase gene from V. philippica (VpPDS) was silenced, and local leaf bleaching was observed but did not exhibit systemic effects, thereby limiting utilization of TRV-mediated gene silencing in the recipient plant. However, we observed systemic gene silencing in Nicotiana benthamiana when the VpPDS sequence was used as a trigger, thereby suggesting that heterologous gene sequences could elicit gene silencing. We then investigated the role of gene PISTILLATA from V. philippica (VpPI) of the B-class MADS-box gene family, which regulates the identity and development of stamens and petals. Using the coding region of VpPI as triggers, we determined the gene silencing efficiency of the corresponding GLOBOSA paralogs in N. benthamiana (NbGLO1 and NbGLO2), and we observed stamen-to-carpel transformation and distorted corollas in N. benthamiana suggesting that VpPI functioned as the NbGLO gene. However, the 3'-untranslated region (3'UTR) of VpPI and each 3'UTR of the NbGLO genes downregulated its own corresponding gene, hardly producing floral homeotic alterations. This work provides a better understanding of gene-specific probe design for gene silencing as well as shows that heterologous sequence-triggered VIGS is an efficient way to investigate functional conservation of orthologous genes.

Salt stress is one of the most critical environmental factors limiting plant growth, and hydrogen sulfide (H2S) can play a role in plant responses to this stress. To investigate the effects of H2S on mitochondrial functions under salt stress, we treated Malus hupehensis Rehd. var. pingyiensis germinating seeds with an 85 mM NaCl solution with or without an H2S donor sodium hydrosulfide (NaHS) and H2S scavenger hypotaurine (HT). Then, hydrogen peroxide (H2O2) content and antioxidant enzyme activities were measured in mitochondria of seedling roots. Our results show that the application of 0.05 mM NaHS rescued an NaCl-induced inhibition of root elongation, decreased H2O2 content, and enhanced superoxide dismutase (SOD), guaiacol peroxidase (POD), and catalase (CAT) activities in the mitochondria compared to NaCl treatment alone. It was also found that 0.05 mM NaHS significantly decreased the mitochondrial permeability transition pore and increased mitochondrial membrane fluidity, mitochondrial membrane potential, and cytochrome c/a ratio under NaCl stress. However, 0.02 mM NaHS did not affect root growth, antioxidant enzyme activities, and mitochondrial function under NaCl stress, whereas high concentrations of NaHS (more than 0.2 mM) had a weaker or negative effects. Moreover, 15 µM HT eliminated the beneficial effects of NaHS under NaCl stress. Our results suggest that H2S protected plants against salt stress by decreasing H2O2 accumulation and by regulating membrane stability and antioxidant system in mitochondria.

Iron availability affects plant growth depending on soil type. Mangroves are characterized by alkaline soils in which the halophytic wild rice relative Porteresia coarctata thrives. Young plants of P. coarctata grew optimally in the presence of 150 mM NaCl in a hydroponic medium and tolerated iron deficiency and salt up to 21 d without showing any symptoms of stress. A homolog of the rice iron deficiency responsive cis-acting element binding factor (IDEF1) that functions at the base of an iron regulated network was isolated and characterized from P. coarctata. PcIDEF1 had a close paralog in P. coarctata genome and its transcript expression was upregulated by both iron deficient conditions and salt treated conditions for up to three weeks. Sub-cellular localization study suggests nuclear targeting PcIDEF1 protein in guard cells and root tissues of tobacco. In vitro assays for metal binding affinity and binding PcIDEF1 to iron deficiency responsive element 1 (IDE1)-like elements in the 5' flanking region of an iron regulated transporter from P. coarctata suggest that PcIDEF1 could potentially sense iron content in a plant cell and regulate expression of iron responsive genes containing IDE1-like elements in their promoter region. This study provides evidence for a possible cross-talk between iron deficiency and salt responses.

Little is known about the contributions of DNA methylation/demethylation to plant aging and senescence. We used Arabidopsis thaliana to study how increasing age of an annual plant species influences DNA methylation. Based on methylation-sensitive DNA fragmentation assay, it could be concluded that aging A. thaliana was accompanied by DNA demethylation. Bisulfite sequencing reveals that cytosine methylation within the Actin2 3' untranslated region and internal transcribed spacer with 5.8S rRNA (ITS1-5.8SrRNA-ITS2) DNA regions decreased with A. thaliana growth and aging. We show that transcription of methyltransferase genes, chromomethyltransferase AtCMT3 and methyltransferse AtMETI, significantly decreased during development and aging of the A. thaliana plants, whereas expression of demethylase genes - repressor of silencing AtROS1, demeter AtDME, and demeter-like AtDML2 and AtDML3 - increased at least at some stages of plant development. The data obtained in the present study suggest that plant DNA regions may undergo demethylation during plant aging via reduction of DNA methylation processes and activation of active DNA demethylation.

Sprouts of potato tubers were excised from the three potato cultivars Agria, Hermes, and Spunta, sterilized and subjected to shoot formation and propagation on Murashige and Skoog (MS) medium supplemented with 1 mg dm^-3 6-benzylaminopurine (BAP) + 0.5 mg dm^-3 gibberellic acid. Shoots were rooted on MS medium supplemented with 1 mg dm^-3 indole-3-butyric acid. To increase shoot vigour prior tuber formation, shoots were subcultured on MS medium supplemented with 0.56 mg dm^-3 BAP, 0.11 mg dm^-3 2,4-dichlorophenoxyacetic acid, and 0.96 mg dm^-3 naphthaleneacetic acid. Under dark, microtuberization on MS media supplemented with 4 mg dm^-3 of both BAP and kinetin was better than 4 mg dm^-3 BAP alone, where they induced higher number of microtubers per shoot and/or the percentage of shoots that formed microtubers. The highest frequency of microtuber formation was achieved when sucrose at high concentration (8 %) was used as carbon source in culture media. Glucose ranked at the second position whereas fructose reduced the microtuber formation frequency when it was used alone or in combination with glucose. Under the applied culture conditions, cvs. Agria and Hermes showed better micropropagation and microtuberization in comparison to cv. Spunta. In addition, isozyme and RAPD techniques revealed that Agria and Hermes are closer to each other when compared with the third cultivar.

About 70 % of the total land area in the world are affected by soil freeze and thaw (FT) cycles. Root is the first organ of plant to sense soil environment and it is unclear how it copes with the soil FT. Based on the different functions of firstorder pioneer and fibrous roots in woody plants, we hypothesize that pioneer and fibrous roots respond differently. The experiment was conducted in a growth chamber using Picea asperata seedlings. We designed the FT based on field observation data. The physiological responses in fibrous and pioneer roots were examined. Fibrous roots had higher root vitality and N content, whereas pioneer roots exhibited higher total nonstructural saccharide content. The accumulation of O₂ ^- under FT treatment was similar in the two types of roots. Pioneer roots showed higher osmolyte (especially proline) content, whereas fibrous roots had higher peroxidase activity. The present study confirmed that fibrous roots have stronger metabolism ability, whereas pioneer roots are the key storage organs. FT in the temperature range from -5 to 5 °C are mild and do not cause serious injury to roots. Pioneer roots have higher tolerance to soil FT in spring than fibrous roots. The roots have different strategies to FT: fibrous roots increase the antioxidant system, whereas pioneer roots accumulate more osmolytes. Such knowledge can help us to understand how roots of woody plants cope with soil FT.

Na⁺/H⁺ exchanger (NHX)-mediated Na⁺ and H⁺ antiport is an important mechanism for salt tolerance in plants. In this study, an Na⁺/H⁺ antiporter gene, referred to as NsNHX1, was isolated from the halophyte Nitraria sibirica Pall. using degenerate polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). The resulting 2 182 bp NsNHX1 cDNA contained a 1 635 bp open reading frame (ORF) that encoded 544 amino acids and showed striking sequence similarity to tonoplast-localized NHXs from other plants. Subcellular localization analysis confirmed NsNHX1 to be a tonoplast-localized protein. Cis-elements described as being responsive to biotic and abiotic stresses were present in the NsNHX1 promoter region, and reverse transcription (RT)-PCR analysis confirmed that NsNHX1 expression was induced by exogenous abscisic acid (ABA), cold, and NaCl. Transcription of NsNHX1 increased sharply 3 h after treatment with 200 mM NaCl revealing that NsNHX1 responded rapidly to the salt stress. Overexpression of NsNHX1 enhanced salt tolerance in transgenic Arabidopsis thalliana L. suggesting that NsNHX1-mediated Na⁺ compartmentalization played an important role in enhancing plant salt tolerance.

The rice dwarf and narrow leaf mutant 2 (dnl2) is dwarfed and forms narrow and brittle leaves. Its dwarfness was shown to be due to its shortened internodes resulting from a reduced size of the internode parenchyma cells. Its narrow and brittle leaves were attributed to a compromised ability to form vascular bundles but a reduced fiber content and thin cortical layer. However, response to the application of either gibberellin or brassinolide was not different between dnl2 and its wild type. Transcription profiling indicates that a number of cell division/expansion-associated and crude fiber synthesis-related genes were down-regulated in the mutant. A genetic analysis revealed that the mutant phenotype was under monogenic control, and the gene responsible was mapped to a 50.1 kb genomic region on the long arm of chromosome 10. This region was shown to harbor 10 open reading frames. Although transcription profiling these genes indicates that three were differentially transcribed in the mutant, there was no sequence polymorphism in the coding sequence between the mutant and the wild type alleles.

The effect of polyamine biosynthesis inhibitors on the salt stress response of rice seedlings was investigated. For this, DL-α-difluoromethylarginine (DFMA) and DL-α-difluoromethylornithine (DFMO), two competitive inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), were used. The ADC and ODC are rate-limiting enzymes involved in synthesis of putrescine. The effective quantum yield of photosynthetic energy conversion (Φ_PSII) decreased with the salt stress, and this decrease was highly significant in the treatments with DFMA and DFMO. Interestingly, addition of exogenous putrescine reduced the decline of Φ_PSII. Putrescine content strongly decreased after one day of the inhibitor treatment. Although the content of spermidine (converted from putrescine) also showed an initial decrease in response to the inhibitors, it recovered to a similar level to that in the control after 3 d of treatment. Under the salt stress, the effect of the inhibitors on the different compounds was similar. Moreover, the addition of exogenous putrescine partially suppressed the decrease in spermidine and spermine content. A positive correlation between the spermidine and spermine content and the Φ_PSII was observed. The results suggest that, under salt stress, a decrease in polyamine biosynthesis and/or polyamine content has a strong negative effect on leaves and increases salt stress sensitivity.

Artemisinin, a natural sesquiterpenoid isolated from Artemisia annua L., is regarded as the most efficient drug against malaria in the world. Artemsinin production in NaCl-treated A. annua seedlings and its relationships with the glucose-6-phosphate dehydrogenase (G6PDH) activity and generation of H₂O₂ and nitric oxide (NO) were investigated. Results revealed that artemisinin content in the seedlings was increased by 79.3 % over the control after 1-month treatment with 68 mM NaCl. The G6PDH activity was enhanced in the presence of NaCl together with stimulated generation of H₂O₂ and NO. Application of 1.0 mM glucosamine (GlcN), an inhibitor of G6PDH, blocked the increase of NADPH oxidase and nitrate reductase (NR) activities, as well as H₂O₂ and NO production in A. annua seedlings under the salt stress. The induced H₂O₂ was found to be involved in the upgrading gene expression of two key enzymes in the later stage of artemisinin biosynthetic pathway: amorphadiene synthase (ADS) and amorpha-4,11-diene monooxygenase (CYP71AV1). The released NO being attributed mainly to the increase of NR activity, negatively interacted with H₂O₂ production and enhanced gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Inhibition of NO generation partly blocked NaCl-induced artemisinin accumulation, and NO donor strongly rescued the decreased content of artemisinin caused by GlcN. These results suggest that G6PDH could play a critical role in NaCl-induced responses and artemisinin biosynthesis in A. annua.

Brassinolide (BL) alleviates salt injury in cotton seedlings; however, little is known about the molecular mechanisms of this response. In this study, digital gene expression analysis was performed to better understand the regulatory pathways of BL in NaCl-stressed cotton (Gossypium hirsutum L.). Compared with control plants (CK), a total of 1 162 and 7 659 differentially expressed genes (DEGs) were detected in the leaves and roots of NaCl-treated plants, respectively. Most of the DEGs in NaCl-treated plants, compared to CK, were regulated by BL. Moreover, expression patterns of DEGs in BL+NaCl treated plants were similar to those in CK plants; however, the responses of DEGs in the leaves and roots of NaCl-treated plants to BL differed. In the roots, BL-regulated DEGs were involved in protein biosynthesis, whereas in the leaves, BL promoted photosynthesis in NaCl-stressed cotton. BL treatment also significantly increased the overall biomass, chlorophyll a + b content in leaves, and the protein content in roots in NaCl-stressed cotton. The downregulation of stress-responsive genes in BL+NaCl-stressed leaves was also found. These results suggest that BL can alleviate NaCl injury in cotton plants.

The present study aimed to investigate the effects of ethanolamine on glycine betaine and proline metabolism in Nicotiana rustica under salt stress. The in vitro grown tobacco (Nicotiana rustica) plants were pretreated with ethanolamine (at concentrations 70, 130, 270, and 530 μM for biochemical analysis and only at the concentration of 530 μM for molecular analysis) and then transferred to Murashige and Skoog medium containing 200 mM NaCl for 3 weeks. Our results showed that ethanolamine promoted glycine betaine biosynthesis by an increase in betaine aldehyde dehydrogenase (BADH) gene expression and BADH enzymatic activity. Moreover, ethanolamine pretreatment possibly reduced proline content in salt stressed plants via its negative effect on Δ-pyrroline-5-carboxylate synthase (P5CS) gene expression and P5CS enzymatic activity and its positive effect on proline dehydrogenase (PDH) gene expression and PDH activity.

The roles of ethylene (ET) or salicylic acid (SA) in plant response to low temperature (LT, 5 °C) have been implicated. However, the combined effect of ET- and SA-signaling on plant growth and metabolism under LT remains to be evaluated. In this study, we comparatively analyzed the response of Arabidopsis ethylene insensitive (ein) 2-1 (an ET insensitive mutant), nonexprressor of pathogenesis relative (npr)1-1 (an SA insensitive mutant) and double mutant ein2-1/npr1-1 plants to LT. The results show that a LT of 5 °C induced plant growth retardation to a less degree in ein2-1, an intermediate degree in npr1-1, but a much larger in ein2-1/npr1-1 compared to the wild-type (WT) plants. The LT susceptibility of the ein2-1/npr1-1 plants was correlated to a lower net photosynthetic rate and proline content, and a higher content of H₂O₂ and malondialdehyde and electrolyte leakage relative to the WT plants. Lower activities of superoxide dismutase, peroxidase, and catalase, as well as a lower glutathione content and a ratio of its reduced form to its oxidized form were also observed in the double mutant plants as compared with the WT plants. However, at normal conditions (23 °C), all the tested physiological and biochemical parameters were comparable between the ein2-1/npr1-1 and WT plants, and plant growth was even better in the double mutant than in the WT plants. On the contrary, most of the above-mentioned parameters were advantageous in the ein2-1 and npr1-1 plants over the WT plants under the LT conditions. These data suggest that a parallel function or physiological redundancy of nonexpressor of pathogenesis relative 1 and ethylene insensitive 2 existed in the Arabidopsis plant response to the LT. On the other hand, an interaction between ET- and SA-signaling occurred during this process.

Root epidermis and apoplastic barriers (endodermis and exodermis) are the critical root structures involved in setting up plant-soil interface by regulating free apoplastic movement of solutes within root tissues. Probing root apoplast permeability with "apoplastic tracers" presents one of scarce tools available for detection of "apoplastic leakage" sites and evaluation of their role in overall root uptake of water, nutrients, or pollutants. Although the tracers are used for many decades, there is still not an ideal apoplastic tracer and flawless procedure with straightforward interpretation. In this article, we present our experience with the most frequently used tracers representing various types of chemicals with different characteristics. We examine their behaviour, characteristics, and limitations. Here, we show that results gained with an apoplastic tracer assay technique are reliable but depend on many parameters-chemical properties of a selected tracer, plant species, cell wall properties, exposure time, or sample processing.

The speciation allele at Eml-A1 of hexaploid wheat, which causes embryo lethality in wheat-rye hybrids, was investigated using cytologically modified genetic stocks. It was demonstrated that an extra dose of this allele had no effect on embryo development in these hybrids. There was no positive effect on embryo development and, therefore, no overcoming of the postzygotic barrier. An abortion of the hybrid embryos at an earlier stage of development was also not observed. Physical mapping was performed using chromosome 6A deletion lines. This study revealed the location of Eml-A1 on the most distal part of the long arm of chromosome 6A. To identify possible candidate genes responsible for embryo lethality, in silico sequence homology analysis was performed. Two candidate genes for Eml-A1 that are involved in shoot apical meristem maintenance were identified on chromosome 6AL. However, functional validation assays need to be designed and performed.

Ran is involved in response to external stimuli. In this study, six MsRan gene cDNA sequences were isolated from wild banana (Musa spp. AB group) from Sanming City, China. Sequence analysis reveals that MsRan3A, MsRan3A-1a, and MsRan3C contained Ran protein domains including a GTP hydrolysis domain, a RanGAP-binding domain, and an acidic tail, whereas two G boxes (G4 and G5) were absent in MsRan3A-6a. The physicochemical property of MsRan3A, MsRan3A-1a, MsRan3A-6a, and MsRan3C appeared to differ significantly. Real time quantitative PCR (qPCR) analysis indicates that MsRan3A-1, MsRan3A-5, MsRan3A-6, MsRan3A-6a, and MsRan3C-1 were expressed in roots, leaves, peduncles, bracts, flowers, peels, and pulp of the wild banana. MsRan3A-1a was expressed at extremely low levels in these tissues and was undetectable by qPCR. The MsRan genes were found to be involved in responses to a low temperature stress but with different response patterns. Furthermore, salicylic acid significantly enhanced MsRan gene expressions suggesting the involvement of these genes in salicylic acid signal transduction.

We used flowering branches at the budding stage of two peach cultivars Xiahui 6 and Xiacui with different cold resistance to explore the effect of exogenous salicylic acid (SA) on the freezing injury of peach floral organs and the molecular mechanism. Using water application as the negative control, the effects of spraying with SA at concentrations of 20 or 100 mg dm^-3 on stigma receptivity, frost damage characteristics of floral organs, and the expressions of C-repeat-binding factor (PpCBF) gene family members were investigated at 0 °C. No significant frost damage was observed on petals in all treatments. No frost damage was seen in the ovary and style under 20 mg dm^-3 SA treatment, but damage was substantial at the other two treatments. Cultivar Xiahui 6 was more susceptible to freezing than cv. Xiacui. The expression peaks of PpCBFs in the SA-pretreated floral organs occurred at 3 or 6 h after low temperature treatment, and peak time was closely related to peach cultivar, organ, and SA concentration. This indicates that appropriate concentration of exogenous SA may alleviate freezing damage to floral organs and enhance cold resistance by the regulated expression pattern of PpCBF.

Phenylalanine ammonia lyase (PAL) is a specific branch point enzyme of primary and secondary metabolism. The Citrus reticulata Blanco PAL gene was cloned and designated as CrPAL1. The cDNA sequence of CrPAL1 was 2 166 bp, encoding 721 amino acid residues. Sequence alignment indicates that CrPAL1 shared a high identity with PAL genes found in other plants. Both the dominant and catalytic active sites of CrPAL1 were similar to PAL proteins observed in Petroselinum crispum. Phylogenetic tree analysis indicates that CrPAL1 was more closely related to PALs in Citrus clementina × C. reticulata and Poncirus trifoliata than to those from other plants. Subcellular localization reveals that CrPAL1-green fluorescent protein fusion protein was specifically localized in the plasma membrane. Activity of PAL as well as CrPAL1 expression increased under Fe deficiency. A similar result was noted for total phenolic content. The root exudates of C. reticulata strongly promoted reutilization of apoplastic Fe in roots. Furthermore, Fe was more desorbed from the cell wall under Fe deficiency than in sufficient Fe supply.

The effects of nitric oxide (NO) and/or iron (Fe) supplied to Fe deficient plants have been investigated in peanut (Arachis hypogaea L.) grown in Hoagland nutrient solution with or without Fe. Two weeks after Fe deprivation, recovery was induced by addition of 250 μM sodium nitroprusside (SNP, a NO donor) and/or 50 μM Fe (Fe-EDTA) to the Fe deprived (-Fe) nutrient solution. Activities of antioxidant enzymes, leaf chlorophyll (Chl), and active Fe content decreased, whereas activities of H⁺-ATPase, ferric-chelate reductase (FCR), nitrate reductase, and nitric oxide synthase and NO production increased in Fe deficient plants, consequently an Fe chlorosis symptom appeared obviously. In contrast, these symptoms disappeared gradually after two weeks with NO and/or Fe supply, which caused an increases in leaf Chl and active Fe content, especially following by co-treatment with NO and Fe to values found in Fe sufficient plants. Increased activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) and decreased accumulation of reactive oxygen species (H₂O₂, O₂^*- ) and malondialdehyde enhanced the ability of resistance to oxidative stress. Supplied NO alone had the obvious effect on increased NO production and on activity of H⁺-ATPase and FCR, whereas root length and root/shoot ratio were most effectively increased by Fe supplied alone. Co-treatment with NO and Fe did the best effects on recovery peanut chlorosis symptoms by significantly increased Chl and available Fe content and adjusted distribution of Fe and other mineral elements (Ca, Mg, and Zn) in both leaves and roots.

ABC transporters, which comprise one of the largest protein families, are involved in maintaining osmotic homeostasis, nutrient uptake, pathogen resistance, and metal tolerance. In this study, 30 ABC genes in dwarf polish wheat were characterized and classified into seven subfamilies (ABCA - ABCG). Among them, 24 ABC transporters were newly found in wheat. The expressions of 13 ABC genes in roots and leaves under six metal stresses were also analyzed. All these genes were differentially regulated by Cd (except ABCE2, ABCF4, and ABCF6 in roots), suggesting that these genes participate in Cd transport, sequestration, or uptake. These genes were also differentially regulated by other metals including Cu, Mg, Zn, Fe, and Ni. Results suggest that the expressions of ABC transporters in dwarf polish wheat played important roles in metal transport and detoxification.

The accumulation of polysaccharides, activities of sucrose metabolism enzymes, and the expression of sucrose biosynthetic genes in Dendrobium officinale were significantly affected by exogenous methyl jasmonate (MeJA). Application of MeJA increased the content of polysaccharides and the highest polysaccharide production occurred in the samples treated with 200 μM MeJa. The MeJA application influenced polysaccharide biosynthesis rather than degradation because the activities of sucrose metabolism enzymes and the expressions of sucrose biosynthetic genes were upregulated by MeJA. Interestingly, low MeJA concentrations promoted accumulation of Dendrobium polysaccharides, while high MeJA amounts played an inhibitory role. The content of major constituent of polysaccharides, glucose and mannose, also increased after MeJa treatment.

Root rot caused by Fusarium solani, is one of the most severe diseases in pepper (Capsicum annuum L.). Grafting has been attempted as an effective means to control the disease, but little is known about the disease resistance mechanism in grafted pepper. Therefore, we investigated the changes of biomass, cell structure, and the secondary metabolism in roots of control (non-grafted pepper) and grafted peppers using cvs. Weishi and Buyeding as rootstocks and the cv. Xinfeng 2 as a scion. After a manual inoculation, less F. solani invaded grafted pepper roots and consequently less serious injury to the root cell ultra-structure compared with the control was found. The roots of grafted pepper infected with F. solani exhibited greater biomass production and root activity than the roots of infected controls. Grafting led to an increased content of salicylic acid, benzoic acid, vanillin, lignin, and polyamines, as well as activities of phenylalanine ammonia lyase, polyphenoloxidase, and peroxidase. These results suggest that grafting improved the resistance of peppers to root rot.