Aquaporins in cereals-important players in maintaining cell homeostasis under abiotic stres

Cereal productivity is reduced by environmental stresses such as drought, heat, elevated CO2, salinity, metal toxicity and cold. Sometimes, plants are exposed to multiple stresses simultaneously. Plants must be able to make a rapid and adequate response to these environmental stimuli in order to restore their growing ability. The latest research has shown that aquaporins are important players in maintaining cell homeostasis under abiotic stress. Aquaporins are membrane intrinsic proteins (MIP) that form pores in the cellular membranes, which facilitate the movement of water and many other molecules such as ammonia, urea, CO2, micronutrients (silicon and boron), glycerol and reactive oxygen species (hydrogen peroxide) across the cell and intercellular compartments. The present review primarily focuses on the diversity of aquaporins in cereal species, their cellular and subcellular localisation, their expression and their functioning under abiotic stresses. Lastly, this review discusses the potential use of mutants and plants that overexpress the aquaporin-encoding genes to improve their tolerance to abiotic stress

TIP Aquaporins in Plants: Role in Abiotic Stress Tolerance

Tonoplast Intrinsic Proteins (TIP) are one of five subfamilies of aquaporins in higher plants. Plants typically contain a large number of TIP genes, ranging from 6 to 35 compared to humans. The molecular weight of the TIP subfamily members ranges from 25 to 28 kDa. Despite their sequence diversity, all TIP monomers have the same structure, which consists of six transmembrane helices and five inter-helical loops that form an hourglass shape with a central pore. Four monomers form tetramers, which are functional units in the membrane. TIPs form channels in the tonoplast that basically function as regulators of the intracellular water flow, which implies that they have a role in regulating cell turgor. TIPs are responsible for precisely regulating the movement of not only water, but also some small neutral molecules such as glycerol, urea, ammonia, hydrogen peroxide and formamide. The expression of TIPs may be affected by different environmental stresses, including drought, salinity and cold. TIPs expression is also altered by phytohormones and the appropriate cis-regulatory motifs are identified in the promotor region of the genes encoding TIPs in different plant species. It was shown that manipulating TIP-encoding genes expression in plants could have the potential to improve abiotic stress tolerance

Drought stress and re-watering affect the abundance of TIP aquaporin transcripts in barley

Tonoplast Intrinsic Proteins (TIP) are plant aquaporins that are primarily localized in the tonoplast and play a role in the bidirectional flux of water and other substrates across a membrane. In barley, eleven members of the HvTIP gene subfamily have been identified. Here, we describe the transcription profile of the HvTIP genes in the leaves of barley seedlings being grown under optimal moisture conditions, drought stress and a re-watering phase. The applied drought stress caused a 55% decrease in the relative water content (RWC) in seedlings, while re-watering increased the RWC to 90% of the control. Our analysis showed that all HvTIP genes, except HvTIP3;2, HvTIP4;3 and HvTIP5.1, were expressed in leaves of ten-day-old barley seedlings under optimal water conditions with the transcripts of HvTIP2;3, HvTIP1;2 and HvTIP1;1 being the most abundant. We showed, for the first time in barley, a significant variation in the transcriptional activity between the analysed genes under drought stress. After drought treatment, five HvTIP genes, which are engaged in water transport, were down-regulated to varying degrees, while two, HvTIP3;1 and HvTIP4;1, were up-regulated. The HvTIP3;1 isoform, which is postulated as transporting hydrogen peroxide, expressed the highest increase of activity (ca. 5000x) under drought stress, thus indicating its importance in the response to this stress. Re-hydration caused the return of the expression of many genes to the level that was observed under optimal moisture conditions or, at least, a change in this direction Additionally, we examined the promotor regions of HvTIP and detected the presence of the cis-regulatory elements that are connected with the hormone and stress responses in all of the genes. Overall, our results suggest that 7 of 11 studied HvTIP (HvTIP1;1, HvTIP1;2, HvTIP2;1, HvTIP2;2, HvTIP2;3, HvTIP3;1, HvTIP4;1) have an important function during the adaptation of barley to drought stress conditions. We discuss the identified drought-responsive HvTIP in terms of their function in the adaptation of barley to this stress

Molecular analysis of point mutations in a barley genome exposed to MNU and Gamma rays

We present studies aimed at determining the types and frequencies of mutations induced in the barley genome after treatment with chemical (N-methyl-N-nitrosourea, MNU) and physical (gamma rays) mutagens. We created M2 populations of a doubled haploid line and used them for the analysis of mutations in targeted DNA sequences and over an entire barley genome using TILLING (Targeting Induced Local Lesions in Genomes) and AFLP (Amplified Fragment Length Polymorphism) technique, respectively. Based on the TILLING analysis of the total DNA sequence of 4,537,117 bp in the MNU population, the average mutation density was estimated as 1/504 kb. Only one nucleotide change was found after an analysis of 3,207,444 bp derived from the highest dose of gamma rays applied. MNU was clearly a more efficient mutagen than gamma rays in inducing point mutations in barley. The majority (63.6%) of the MNU-induced nucleotide changes were transitions, with a similar number of G > A and C > T substitutions. The similar share of G > A and C > T transitions indicates a lack of bias in the repair of O6-methylguanine lesions between DNA strands. There was, however, a strong specificity of the nucleotide surrounding the O6-meG at the −1 position. Purines formed 81% of nucleotides observed at the −1 site. Scanning the barley genome with AFLP markers revealed ca. a three times higher level of AFLP polymorphism in MNUtreated as compared to the gamma-irradiated population. In order to check whether AFLP markers can really scan the whole barley genome for mutagen-induced polymorphism, 114 different AFLP products, were cloned and sequenced. 94% of bands were heterogenic, with some bands containing up to 8 different amplicons. The polymorphic AFLP products were characterised in terms of their similarity to the records deposited in a GenBank database. The types of sequences present in the polymorphic bands reflected the organisation of the barley genome

Acromegaly in a patient with normal pituitary gland and somatotropic adenoma located in the sphenoid sinus

Ectopic acromegaly is a very rare clinical entity occurring in less than 1% of acromegalic patients. In most cases it is caused by GHRH or rarely GH&#8211;secreting neoplasms. Even rarer are ectopic pituitary adenomas located in the sphenoid sinus or nasopharynx that originate from pituitary remnants in the craniopharyngeal duct. This dissertation presents the difficulties in visualizing GH-secreting adenoma located in the sphenoid sinus. A 55-year-old man had somatic features of acromegaly for several years. MRI imaging revealed a slightly asymmetric pituitary gland (14 &#165; 4 mm) without focal lesions. Simultaneously, a spherical mass, 10 mm in diameter, corresponding with ectopic microadenoma was demonstrated on the upper wall of the sphenoid sinus. The serum GH level was 4.3 mg/l, IGF-1 = 615 mg/l, and a lack of GH suppression with oral glucose was proven. After preliminary treatment with a long-acting somatostatin analogue, transsphenoidal pituitary tumour removal was performed. Histopathological, electron microscopical and immunohistochemical analysis revealed densely granulated somatotropic pituitary adenoma: GH(+), PRL(-), ACTH(-), TSH(-), FSH(-), LH(-), MIB1 < 1%, SSTR3(+) and SSTR5(+). Post-surgical evaluation showed normal pituitary MRI scans, GH and IGF-1 levels 0.18 &#956;g/l and 140 mg/l, respectively, as well as normal GH suppression with oral glucose. The careful analysis of possible pituitary embryonic malformations points out their significance for proper localization of extrapituitary adenomas.Ektopowa postać akromegalii występuje u mniej niż 1% chorych. W większości przypadków jest ona spowodowana wydzielaniem GHRH lub sporadycznie GH przez guz nowotworowy. Jeszcze rzadsze są ektopowo położone gruczolaki przysadki. Zlokalizowane najczęściej w zatoce klinowej lub nosogardzieli, rozwijają się z fragmentu przysadki pozostałego w przewodzie czaszkowo-gardłowym. Prezentowana praca ukazuje trudności w wizualizacji gruczolaka somatotropowego położonego w zatoce klinowej. Mężczyzna w wieku 55 lat z somatycznymi cechami akromegalii od kilkunastu lat. W MRI przysadka nieco asymetryczna, 14 &#165; 4 mm, bez zmian ogniskowych. Na górnej ścianie zatoki klinowej obecny kulisty obszar o średnicy 10 mm odpowiadający mikrogruczolakowi. Stężenie GH = 4,3 &#956;g/l, IGF-1 = 615 &#956;g/l, brak hamowania GH glukozą. Po przygotowaniu długodziałającym analogiem somatostatyny guz usunięto z dostępu przez zatokę klinową. Badania histopatologiczne, elektronomikroskopowe oraz immunohistochemiczne wykazały obecność bogatoziarnistego somatotropowego gruczolaka przysadki: GH(+), PRL(-), ACTH(-), TSH(-), FSH(-), LH(-), MIB1 < 1%, SSTR3(+), SSTR5(+). W ocenie pooperacyjnej: prawidłowy obraz przysadki w MRI, GH = 0,18 &#956;g/l; IGF-1 = 140 &#956;g/l, obecne hamowanie GH glukozą. Staranna analiza możliwych zaburzeń rozwoju embrionalnego przysadki potwierdza ich znaczenie dla prawidłowej lokalizacji pozaprzysadkowo położonych gruczolaków

Methyl Jasmonate Affects Photosynthesis Efficiency, Expression of HvTIP Genes and Nitrogen Homeostasis in Barley

Jasmonates modulate many growth and developmental processes and act as stress hormones that play an important role in plant tolerance to biotic and abiotic stresses. Therefore, there is a need to identify the genes that are regulated through the jasmonate signalling pathway. Aquaporins, and among them the Tonoplast Intrinsic Proteins (TIPs), form the channels in cell membranes that are responsible for the precise regulation of the movement of water and other substrates between cell compartments. We identified the cis-regulatory motifs for the methyl jasmonate (MeJA)-induced genes in the promoter regions of all the HvTIP genes, which are active in barley seedlings, and thus we hypothesised that the HvTIP expression could be a response to jasmonate signalling. In the presented study, we determined the e ect of methyl jasmonate on the growth parameters and photosynthesis e ciency of barley seedlings that had been exposed to di erent doses of MeJA (15–1000 M 120 h) in a hydroponic solution. All of the applied MeJA concentrations caused a significant reduction of barley seedling growth, which was most evident in the length of the first leaf sheath and dry leaf weight. The observed decrease of the PSII parameters after the exposure to high doses of MeJA (500 M or higher) was associated with the downregulation of HvPsbR gene encoding one of the extrinsic proteins of the Oxygen Evolving Complex. The reduced expression of HvPsbR might lead to the impairment of the OEC action, manifested by the occurrence of the K-band in an analysis of fluorescence kinetics after MeJA treatment as well as reduced photosynthesis e ciency. Furthermore, methyl jasmonate treatment caused a decrease in the nitrogen content in barley leaves, which was associated with an increased expression the four tonoplast aquaporin genes (HvTIP1;2, HvTIP2;2, HvTIP4;1 and HvTIP4;2) predicted to transport the nitrogen compounds from the vacuole to the cytosol. The upregulation of the nitrogen-transporting HvTIPs might suggest their involvement in the vacuolar unloading of ammonia and urea, which both could be remobilised when the nitrogen content in the leaves decreases. Our research provides tips on physiological role of the individual TIP subfamily members of aquaporins under methyl jasmonate action

Barley ABI5 (Abscisic Acid INSENSITIVE 5) Is Involved in Abscisic Acid-Dependent Drought Response

ABA INSENSITIVE 5 (ABI5) is a basic leucine zipper (bZIP) transcription factor which acts in the abscisic acid (ABA) network and is activated in response to abiotic stresses. However, the precise role of barley (Hordeum vulgare) ABI5 in ABA signaling and its function under stress remains elusive. Here, we show that HvABI5 is involved in ABAdependent regulation of barley response to drought stress. We identified barley TILLING mutants carrying different alleles in the HvABI5 gene and we studied in detail the physiological and molecular response to drought and ABA for one of them. The hvabi5.d mutant, carrying G1751A transition, was insensitive to ABA during seed germination, yet it showed the ability to store more water than its parent cv. “Sebastian” (WT) in response to drought stress. The drought-tolerant phenotype of hvabi5.d was associated with better membrane protection, higher flavonoid content, and faster stomatal closure in the mutant under stress compared to the WT. The microarray transcriptome analysis revealed up-regulation of genes associated with cell protection mechanisms in the mutant. Furthermore, HvABI5 target genes: HVA1 and HVA22 showed higher activity after drought, which may imply better adaptation of hvabi5.d to stress. On the other hand, chlorophyll content in hvabi5.d was lower than in WT, which was associated with decreased photosynthesis efficiency observed in the mutant after drought treatment. To verify that HvABI5 acts in the ABA-dependent manner we analyzed expression of selected genes related to ABA pathway in hvabi5.d and its WT parent after drought and ABA treatments. The expression of key genes involved in ABA metabolism and signaling differed in the mutant and the WT under stress. Droughtinduced increase of expression of HvNCED1, HvBG8, HvSnRK2.1, and HvPP2C4 genes was 2–20 times higher in hvabi5.d compared to “Sebastian”. We also observed a faster stomatal closure in hvabi5.d and much higher induction of HvNCED1 and HvSnRK2.1 genes after ABA treatment. Together, these findings demonstrate that HvABI5 plays a role in regulation of drought response in barley and suggest that HvABI5 might be engaged in the fine tuning of ABA signaling by a feedback regulation between biosynthetic and signaling events. In addition, they point to different mechanisms of HvABI5 action in regulating drought response and seed germination in barley

Is it the end of TILLING era in plant science?

Since its introduction in 2000, the TILLING strategy has been widely used in plant research to create novel genetic diversity. TILLING is based on chemical or physical mutagenesis followed by the rapid identification of mutations within genes of interest. TILLING mutants may be used for functional analysis of genes and being nontransgenic, they may be directly used in pre-breeding programs. Nevertheless, classical mutagenesis is a random process, giving rise to mutations all over the genome. Therefore TILLING mutants carry background mutations, some of which may affect the phenotype and should be eliminated, which is often time-consuming. Recently, new strategies of targeted genome editing, including CRISPR/Cas9-based methods, have been developed and optimized for many plant species. These methods precisely target only genes of interest and produce very few off-targets. Thus, the question arises: is it the end of TILLING era in plant studies? In this review, we recap the basics of the TILLING strategy, summarize the current status of plant TILLING research and present recent TILLING achievements. Based on these reports, we conclude that TILLING still plays an important role in plant research as a valuable tool for generating genetic variation for genomics and breeding projects

Barley primary microRNA expression pattern is affected by soil water availability

MicroRNAs are short molecules of 21–24 nt in length. They are present in all eukaryotic organisms and regulate gene expression by guiding posttranscriptional silencing of mRNAs. In plants, they are key players in signal transduction, growth and development, and in response to abiotic and biotic stresses. Barley (Hordeum vulgare) is an economically important monocotyledonous crop plant. Drought is the world’s main cause of loss in cereal production. We have constructed a highthroughput Real-Time RT-qPCR platform for parallel determination of 159 barley primary microRNAs’ levels. The platform was tested for two drought-and-rehydrationtreated barley genotypes (Rolap and Sebastian). We have determined changes in the expression of primary microRNAs responding to mild drought, severe drought, and rehydration. Based on the results obtained, we conclude that alteration in the primary microRNA expression is relative to the stress’s intensity. Mild drought and rehydration mostly decrease the pri-miRNA levels in both of the tested genotypes. Severe drought mainly induces the primary microRNA expression. The main difference between the genotypes tested was a much-stronger induction of pri-miRNAs in Rolap encountering severe drought. The primary microRNAs respond dynamically to mild drought, severe drought, and rehydration treatments. We propose that some of the individual primiRNAs could be used as drought stress or rehydration markers. The usage of the platform in biotechnology is also postulated

Creation of a TILLING population in barley after chemical mutagenesis with sodium azide and MNU

Since the development of the Targeting Induced Local Lesions in Genome (TILLING) strategy, it has been applied in both plants and animals in many studies. The creation of an appropriate population is the first and most crucial step of TILLING. The goal is to obtain a highly mutagenized population that allows many mutations in any gene of interest to be found. Therefore, an effective method of mutation induction should be developed. A high mutation density is associated with saving time, costs, and the labor required for the development of a TILLING platform. The proper handling of the mutated generations, the establishment of a seed bank, and the development of a DNA library are essential for creating a TILLING population. The database in which all of the data from the molecular and phenotypic analyses are collected is a very useful tool for maintaining such population. Once developed, a TILLING population can serve as a renewable resource of mutations for research that uses both forward and reverse genetic approaches. In this chapter, we describe the methods for the development and maintenance of a TILLING population in barley