Physiological and iTRAQ-based quantitative proteomics analyses reveal the similarities and differences in stress responses between short-term boron deficiency and toxicity in wheat roots.

BACKGROUND: Boron (B) is a trace element that is essential for normal wheat development, such as root growth. In wheat, roots are important organs that absorb nutrients and water. However, at present, there is insufficient research on the molecular mechanism underlying how short-term B stress affects wheat root growth. METHODS AND RESULTS: Here, the optimal concentration of B for wheat root growth was determined, and the proteomic profiles of roots under short-term B deficiency and toxicity were analyzed and compared by the isobaric tag for relative and absolute quantitation (iTRAQ) technique. A total of 270 differentially abundant proteins (DAPs) that accumulated in response to B deficiency and 263 DAPs that accumulated in response to B toxicity were identified. Global expression analysis revealed that ethylene, auxin, abscisic acid (ABA), and Ca2+ signals were involved in the responses to these two stresses. Under B deficiency, DAPs related to auxin synthesis or signaling and DAPs involved in calcium signaling increased in abundance. In striking contrast, auxin and calcium signals were repressed under B toxicity. Twenty-one DAPs were detected under both conditions, including RAN1 that played a core role in the auxin and calcium signals. Overexpression of RAN1 was shown to confer plant resistance to B toxicity by activating auxin response genes, including TIR and those identified by iTRAQ in this research. Moreover, growth of the primary roots of tir mutant was significantly inhibited under B toxicity. CONCLUSION: Taken together, these results indicate that some connections were present between RAN1 and the auxin signaling pathway under B toxicity. Therefore, this research provides data for improving the understanding of the molecular mechanism underlying the response to B stress.

The carboxy terminal transmembrane domain of SPL7 mediates interaction with RAN1 at the endoplasmic reticulum to regulate ethylene signalling in Arabidopsis.

In Arabidopsis, copper (Cu) transport to the ethylene receptor ETR1 mediated using RAN1, a Cu transporter located at the endoplasmic reticulum (ER), and Cu homeostasis mediated using SPL7, the key Cu-responsive transcription factor, are two deeply conserved vital processes. However, whether and how the two processes interact to regulate plant development remain elusive. We found that its C-terminal transmembrane domain (TMD) anchors SPL7 to the ER, resulting in dual compartmentalisation of the transcription factor. Immunoprecipitation coupled mass spectrometry, yeast-two-hybrid assay, luciferase complementation imaging and subcellular co-localisation analyses indicate that SPL7 interacts with RAN1 at the ER via the TMD. Genetic analysis revealed that the ethylene-induced triple response was significantly compromised in the spl7 mutant, a phenotype rescuable by RAN1 overexpression but not by SPL7 without the TMD. The genetic interaction was corroborated by molecular analysis showing that SPL7 modulates RAN1 abundance in a TMD-dependent manner. Moreover, SPL7 is feedback regulated by ethylene signalling via EIN3, which binds the SPL7 promoter and represses its transcription. These results demonstrate that ER-anchored SPL7 constitutes a cellular mechanism to regulate RAN1 in ethylene signalling and lay the foundation for investigating how Cu homeostasis conditions ethylene sensitivity in the developmental context.

An Arabidopsis nonhost resistance gene, IMPORTIN ALPHA 2 provides immunity against rice sheath blight pathogen, Rhizoctonia solani.

There is neither resistant rice cultivar nor any control measure against Rhizoctonia solani AG-1 IA (RS), causal of sheath blight and a major threat to global rice production. Rice is a host and Arabidopsis is a nonhost with underlying nonhost resistance (NHR) gene which is largely untested. Using approaches of forward genetics and tools, cytology, and molecular biology, we identified homozygous mutants in Arabidopsis, mapped the NHR gene, and functionally characterized it in response to RS. Rss1 was mapped on Ch 4 between JAERI18 and Ch4_9.18 (844.6 Kb) and identified IMPORTIN ALPHA 2 as the candidate RSS1 gene. We found that breach of immunity in rss1 by RS activates defense responses whereas photosynthetic pigment biosynthesis and developmental processes are negatively regulated. In addition, a gradual decrease in PR1 by 3 dpi revealed that RSS1 positively regulated early SA-mediated resistance. Whereas increased expression of PDF1.2 by 3 dpi supported switching to necrotrophy, SA-mediated defense in Col-0 leading to immune response. Enhanced expression of ATG8a in rss1 supported autophagic cell death. IMPA2, IMPA1, and RAN1 function together to provide NHR against RS. These findings demonstrate that IMPA2 provides NHR against RS in Col-0 that evoke SA-mediated early immunity with boulevard for potential biotechnological application.

Novel insights into the transfer routes of the essential copper cofactor to the ethylene plant hormone receptor family.

The plant hormone ethylene is a key regulator of growth, development and stress adaptation at all stages of the plant life cycle. Signal perception and response to the plant hormone are mediated by a family of receptor kinases localized at the ER-Golgi network which gain their high affinity and specificity for the chemically simple ethylene molecule by an essential copper cofactor bound at their transmembrane domain. Transfer of this cofactor from the plant plasma membrane to the ER-localized receptors requires secured cellular transport of the reactive transition metal. In a recent study, we disclosed the transport proteins involved in the copper transfer to the receptors and identified that cytoplasmic chaperones of the ATX1-family and a membrane-bound P-type ATPase are involved in copper routing. Strictly speaking, our data show that receptors can acquire their copper load by different routes and adopt the metal ion from the plasma membrane either by sequential transfer from soluble chaperones of the ATX1-family via the ER-bound copper-transporting ATPase RAN1 or by direct transfer from the soluble chaperones. Here, we have studied the properties of the soluble plant copper chaperone isoforms, ATX1 and CCH, in more detail. Our data support different cellular functions of these isoforms on copper mobilization.

[Inducible Expression of Ran1 and Its GDP- and GTP-Bound Mimetic Mutants Leads to Defects in Amitosis and Cytokinesis with Abnormal Cytoplasmic Microtubule Assembly].

Ran is an evolutionarily conserved GTPase crucial in regulating various cell divisions, including mitosis and meiosis. A previous study showed that the knockdown of RAN1 inhibited macronuclear amitosis with the abnormal organization of intramacronuclear microtubules in Tetrahymena thermophila. This study aimed to further investigate the effects of the inducible expression of wild-type Ran1 (Ran1WT), GTP-bound Ran1-mimetic (Ran1Q70L), and GDP-bound Ran1-mimetic (Ran1T25N) on cytoplasmic microtubule assembly during amitosis of T. thermophila, based on previous studies about their effects on intramacronuclear microtubule. The mutant strains of T. thermophila for inducible expression of Ran1WT/T25N/Q70L by Cd^(2+) were constructed. The inducibly expressed HA-Ran1Q70L/T25N distributed asymmetrically across the macronuclear envelope during amitosis. At the lower level of inducible expression, only Ran1T25N showed a significant decreasing effect on T. thermophila reproduction, macronuclear amitosis and cytokinesis. At the higher level of inducible expression, Ran1WT/Q70L/T25N inhibited T. thermophila reproduction, macronuclear amitosis and cytokinesis, and the inhibitive effect of Ran1T25N was the most significant. The inducible expression of Ran1WT/Q70L/T25N led to defects in amitosis and cytokinesis with abnormal cytoplasmic microtubule assembly. These results further confirmed the regulatory function of Ran1 on amitosis and suggested a novel role of Ran1 in cytokinesis and the alignment of cytoplasmic microtubules in T. thermophila.

Overexpressing components of the nuclear transport apparatus causes severe growth symptoms in tobacco leaves.

Regulating nucleo-cytoplasmic transport of RNA and protein is a key cellular control point. Perturbing the function of plant nuclear transport components can cause significant developmental defects and in this report we add an important line to this evidence. Overexpression of AtRAN1 or AtNUP62 in Nicotiana benthamiana causes significant damage to leaf tissue. This demonstrates that the precise control of nuclear transport is an important aspect of maintaining tissue integrity.

Arabidopsis RAN1 mediates seed development through its parental ratio by affecting the onset of endosperm cellularization.

Although previous studies have demonstrated that endosperm development is influenced by its parental genome constitution, the genetic basis and molecular mechanisms that control parent-of-origin effects require further elucidation. Here we show that the Ras-related nuclear protein 1 (RAN1) regulates endosperm development in Arabidopsis thaliana. Reciprocal crosses between wild-type (WT) and transgenic lines misexpressing RAN1 (msRAN1) gave rise to small F1 seeds when RAN1 down-regulated/up-regulated individuals were used as a male/female parent; in contrast, F1 seeds were aborted when RAN1 down-regulated/up-regulated plants were used as a female/male parent, suggesting that seed development is affected by the parental genome ratio of RAN1. Whereas RAN1 expression in wild-type plants is reduced before the onset of endosperm cellularization, F1 seeds from reciprocal crosses between WT and msRAN1 showed abnormal endosperm cellularization and ectopic expression of RAN1. The expression of MINISEED3 (MINI3)-a gene that also controls endosperm cellularization-was also affected in these reciprocal crosses, and the misregulation of MINI3 activity rescued F1 seeds when msRAN1 plants were used in reciprocal crosses. Taken together, our results suggest that the parental ratio of RAN1 regulates the onset of endosperm cellularization through its genetic interaction with MINI3.

Current understanding on ethylene signaling in plants: the influence of nutrient availability.

The plant hormone ethylene is involved in many physiological processes, including plant growth, development and senescence. Ethylene also plays a pivotal role in plant response or adaptation under biotic and abiotic stress conditions. In plants, ethylene production often enhances the tolerance to sub-optimal environmental conditions. This role is particularly important from both ecological and agricultural point of views. Among the abiotic stresses, the role of ethylene in plants under nutrient stress conditions has not been completely investigated. In literature few reports are available on the interaction among ethylene and macro- or micro-nutrients. However, the published works clearly demonstrated that several mineral nutrients largely affect ethylene biosynthesis and perception with a strong influence on plant physiology. The aim of this review is to revisit the old findings and recent advances of knowledge regarding the sub-optimal nutrient conditions on the effect of ethylene biosynthesis and perception in plants. The effect of deficiency or excess of the single macronutrient or micronutrient on the ethylene pathway and plant responses are reviewed and discussed. The synergistic and antagonist effect of the different mineral nutrients on ethylene plant responses is critically analyzed. Moreover, this review highlights the status of information between nutritional stresses and plant response, emphasizing the topics that should be further investigated.

Assessment of reference gene stability influenced by extremely divergent disease symptoms in Solanum lycopersicum L.

Tomato (Solanum lycopersicum L.) is one of the most important vegetables of great worldwide economic value. The scientific importance of the vegetable results from the fact that the genome of S. lycopersicum has been sequenced. This allows researchers to study fundamental mechanisms playing an essential role during tomato development and response to environmental factors contributing significantly to cell metabolism alterations. Parallel with the development of contemporary genetics and the constant increase in sequencing data, progress has to be aligned with improvement of experimental methods used for studying genes functions and gene expression levels, of which the quantitative polymerase chain reaction (qPCR) is still the most reliable. As well as with other nucleic acid-based methods used for comparison of the abundance of specific RNAs, the RT-qPCR data have to be normalised to the levels of RNAs represented stably in a cell. To achieve the goal, the so-called housekeeping genes (i.e., RNAs encoding, for instance, proteins playing an important role in the cell metabolism or structure maintenance), are used for normalisation of the target gene expression data. However, a number of studies have indicated the transcriptional instability of commonly used reference genes analysed in different situations or conditions; for instance, the origin of cells, tissue types, or environmental or other experimental conditions. The expression of ten common housekeeping genes of S. lycopersicum, namely EF1α, TUB, CAC, EXP, RPL8, GAPDH, TBP, ACT, SAND and 18S rRNA were examined during viral infections of tomato. Changes in the expression levels of the genes were estimated by comparison of the non-inoculated tomato plants with those infected with commonly known tomato viral pathogens, Tomato torrado virus, Cucumber mosaic virus, Tobacco mosaic virus and Pepino mosaic virus, inducing a diverse range of disease symptoms on the common host, ranging from mild leaves chlorosis to very severe stem necrosis. It is emphasised that despite the wide range of diverse disease symptoms it is concluded that ACT, CAC and EF1α could be used as the most suitable reference genes in studies of host-virus interactions in tomato.