Phylogenetic analysis has become a common step in characterization of gene and protein sequences. However, despite the availability of numerous affordable and more-or-less intuitive software tools, construction of biologically relevant, informative phylogenetic trees remains a process involving several critical steps that are inherently non-algorithmic, i.e., dependent on decisions made by the user. These steps involve, but are not limited to, setting the aims of the phylogenetic study, choosing sequences to be analyzed, and selecting methods employed in sequence alignment construction, as well as algorithms and parameters used to construct the actual phylogenetic tree. This review aims towards providing guidance for these decisions, as well as illustrating common pitfalls and problems occurring during phylogenetic analysis of plant gene sequences.

Kochia sieversiana (Pall.) C.A. Mey. is a forage plant that can grow in extremely alkalinized grasslands at pH 10 or higher. Accumulation of a large amount of oxalic acid (OxA) is a primary characteristic of K. sieversiana. In our study, seedlings of K. sieversiana were exposed to the following conditions: non-stress, salinity (200 mM, a molar ratio of NaCl and Na₂SO₄ 1:1), and alkali stress (200 mM, a molar ratio of NaHCO₃ and Na₂CO₃ 1:1). Growth, water content, content of organic acids (including OxA), Na⁺, and K⁺, and activities of some OxA metabolism-related enzymes were determined. Results show that glycolate oxidase was the key enzyme for OxA synthesis; however, the carboxylation of phosphoenolpyruvate (PEP) by PEP carboxylase (PEPC) probably played a minor role in the OxA-synthetic pathway. The pathway of L-ascorbic acid catabolism was not the main source of OxA accumulation, and the activity of oxalate oxidase (OxO) involved in OxA decomposition was not a limiting factor for inner OxA accumulation. Taken together, accumulation of a large amount of OxA are not related to the degradation and secretion function of OxO but largely depend upon its synthetic function.

SnRK2 are plant-specific serine/threonine kinases that are involved in plant responses to abiotic stresses. In this study, four novel genes SnRK2s:TpSnRK2.11, TpSnRK2.2, TpSnRK2.5, and TpSnRK2.10 from dwarf Polish wheat (Triticum polonicum L.) were characterized and classified into three groups. TpSnRK2.5 and TpSnRK2.11 were members of group 1; TpSnRK2.2 was member of group 2; TpSnRK2.10 belonged to group 3. The expression of TpSNRK2.2 was strongly regulated by polyethylene glycol (PEG), NaCl, and cold in roots and leaves, as well as by ABA in leaves. The transcript of TpSnRK2.5 was intensely induced by all the treatments in roots and leaves. The distinct expression patterns of TpSnRK2.10 indicate that this gene was very sensitive to ABA and NaCl, less sensitive to cold and PEG. The transcript of TpSnRK2.11 was activated significantly by PEG, NaCl, and cold, but weakly by ABA. Our results indicate that these four genes were probably involved in wheat responses to different abiotic stresses in different tissues.

Several studies have been performed to elucidate the role of brassinosteroids (BRs) in plant growth and development. However, information on the role of BR signaling in nutrient uptake is limited. This study explores the relationship between BRs and ammonium transporter 1 (AMT1) expression in Arabidopsis roots. We found that BR treatment reduced the expression of AMT1 genes and that a BR receptor BRI1 mutant bri1-5 reversed its BR-repressed expression. Furthermore, the BR signaling transcription factor, BES1, regulates AMT1 expression in roots. NH₄ ⁺-mediated repression of AMT1;1, AMT1;2, and AMT1;3 was suppressed in a gain-of-function BES1 mutant (bes1-D). This mutant was more sensitive to methyl-ammonium and contained a higher ammonium content compared to wild-type plants. However, BES1 failed to bind E-box elements present in the promoter region of the AMT1 genes. Furthermore, NH₄ ⁺-mediated glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) gene expressions were partially inhibited, and GS activity was slightly lower in the bes1-D mutant relative to that observed in wild-type En2 roots. NH₄ ⁺-mediated AMT1 suppressions are known to be caused by N-metabolites rather than NH₄ ⁺ itself, and glutamine application inhibited AMT1 expression in both En2 and bes1-D indicating that BES1 activation inhibited NH₄ ⁺-mediated GS/GOGAT induction, which might in turn inhibit AMT1 repression. In conclusion, the present study demonstrates that BR regulated nitrogen uptake and assimilation via the BR signaling pathway.

Saussurea involucrata Kar. et Kir. tolerates severe abiotic stresses including cold, and the level of membrane fatty acid desaturation is associated with its cold acclimation. We discovered and characterized a full-length cDNA of stearoyl acyl-carrier-protein desaturase (sikSACPD) which encodes a protein consisted of 396 amino acids. A sequence alignment of the SikSACPD protein showed that it shares 91 and 86 % identity with the SACPDs of Carthamus tinctorius and Helianthus annuus, respectively. Semi-quantitative RT-PCR showed that the expression of sikSACPD increased in S. involucrata leaves as the temperature decreased from 20 to -10 °C. Agrobacterium tumefaciens was used to transform fatty acid biosynthesis 2 (FAB2):SikSACPD and FAB2:FAB2 constructs into tobacco to investigate resistance to a freezing stress and fatty acid composition of the transgenic plants. The FAB2:SikSACPD transgenic plants showed a slightly more resistance to the freezing stress than the FAB2:FAB2 transgenic plants and the wild-type. The proportion of oleic acid (C18:1) in the leaves of SikSACPD transgenic tobacco increased from approximately 5 to 20 % compared with the leaves of non-transgenic tobacco when both were exposed to cold stress treatments. This study demonstrates that the SikSACPD transgene, when expressed in tobacco, conferred a higher cold tolerance in comparison with that observed in non-transgenic tobacco. Thus, this gene may be a candidate for enhancing cold tolerance in other crop plants.

Flow-sorted chromosomes have been used to simplify analyses of complex plant genomes. In bread wheat, majority of studies involve cultivar Chinese Spring, a genotype chosen for sequencing. Telosomic lines developed from this cultivar enable isolation by flow sorting chromosome arms, which represent less than 3.4 % of the genome. However, access to other wheat cultivars is needed to allow mapping and cloning useful genes. In these cultivars, cytogenetic stocks are not readily available and only one chromosome (3B) can be sorted. Remaining chromosomes form composite peaks on flow karyotypes and cannot be sorted. In order to overcome this difficulty, we tested a pragmatic approach in which composite chromosome peaks are dissected to smaller sections. The analysis of chromosome composition in sorted fractions confirmed feasibility of obtaining sub-genomic fractions comprising only a few chromosomes. Usually one of the chromosomes was more abundant and the frequencies of dominant chromosomes in sorted fractions ranged from 16 % (chromosome 7B) to 80 % (chromosome 2B). The enrichment factor, calculated as the relative proportion of chromosomal DNA in the wheat genome to the proportion of chromosomal DNA in a sorted fraction, ranged from 3.2-fold (7B) to 16.4-fold (5D). At least a 5-fold enrichment can be obtained for 17 out of 21 wheat chromosomes. Moreover, we show that 15 out of the 21 chromosomes can be sorted without being contaminated by their homoeologs. These observations provide opportunities for constructing sub-genomic large-insert DNA libraries, optical mapping, and targeted sequencing selected genome regions in various cultivars of wheat. The availability of fractions enriched for chromosomes of interest and free of contaminating homoeologs will increase the efficiency of research projects and reduce their costs as compared to whole genome approaches. The same methodology should be feasible in other plants where single chromosome types cannot be sorted.

Autophagy has been implicated as a cellular protein degradation process that is used to recycle cytoplasmic components under biotic and abiotic stresses and so restrict programmed cell death (PCD). In this study, we report a novel regulatory mechanism by which NADPH oxidase respiratory burst oxidase homolog D (RBOHD) regulated pathogen-induced autophagy and hypersensitive (HR) cell death. We found that the Pseudomonas syringae pv tomato bacteria DC₃000 expressing avrRps4 (Pst-avrRps4) induction of RBOHD-dependent reactive oxygen species (ROS) production promoted the onset of autophagy, whereas a pretreatment with an NADPH oxidase RBOHD inhibitor reversed this trend. The inhibitor significantly blocked pathogen-induced autophagosome formation and ROS increase. Moreover, we also show that in the wild-type and atrbohF mutant, Pst-avrRps4-induced cell death was limited, whereas in the case of the atrbohD mutant, the infection triggered a spreading-type necrosis. Our results demonstrate that the RBOHD-dependent ROS accumulation stimulated autophagosome formation and limited HR cell death.

Assam tea (Camellia assamica) is perennial crop susceptible to moisture stress. We used its tender roots to construct a cDNA library for the identification, functional annotation, and analysis of transcripts. A total of 811 full-length expressed sequence tags were generated. After processing and assembly, 207 unigenes comprising 58 contigs and 149 singletons were registered. Finally, 35.75 % of the unigenes could be assigned to functional categories based on the Arabidopsis proteome. There was 43 % of a coding GC content and 1 272 coding DNA sequences found in the unigenes. Codon usage analysis shows leucine as the highest (9.92 %) and tryptophan (2.0 %) as the lowest coded amino acids. Further, a comparative study with drought-induced genes of young roots (reported earlier) reveals that 4.83 % of genes required for normal growth of roots were also induced by a drought stress. Expressions of 10 unigenes under different abiotic stresses, such as drought, cold, and salinity, were further confirmed by RT-qPCR. The sequence tags generated in this study will be valuable resources for functional genomics study of tea and other woody crop plants in future.

MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Petunia hybrida 'Dainty Lady' employing isopentenyl transferase (ipt) gene as the selection marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. Infected explants were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGR) and antibiotics. Shoots showing extreme shooty phenotype (ESP) were produced from the adventitious shoots separated from the explants. Visual selection was carried out until production of morphologically normal shoots (approximately 4 months after infection). Histochemical GUS assay detected GUS gene in both ESP and normal shoots. PCR analysis confirmed the presence of model gene (GUS gene) and excision of the selection marker (ipt) gene in the normal transgenic plants. The insertion sites (1-3 for ipt gene and 1-2 for GUS gene) were detected by Southern blot analysis using DIG-labeled probes of both genes. These results show that ipt-type MAT vector can be used successfully to produce marker-free transgenic Petunia hybrida plants on PGR- and antibiotic-free MS medium.

The role of auxins on root system architecture was studied by applying indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthaleneacetic acid (NAA) to maize roots and analysing the main processes involved in root development: primary root (PR) elongation, lateral root (LR) formation, and LR root elongation. We found that these effects were not dependent only on concentration, but also on the type of auxin applied. We also studied temporal changes in auxin inhibition of PR elongation. These temporal changes were analysed calculating the elongation ratio between two consecutive one day periods after auxin application. It was observed that a reduction in root elongation was also dependent on the type of auxin applied and its concentration. The inhibitory effect of IBA and IAA decreased on the second day, and the ratio also increased with the concentration. In contrast, NAA increased root elongation inhibition with time. Indeed, the ratio decreased as the NAA concentration increased. Regarding LR formation, we observed that external auxin increased only LR formation in certain zones of the PR. Finally, comparison of inhibition elongation associated with auxin in the LR and PR clearly demonstrates that PR elongation was more sensitive to auxin than LR elongation.

The effects of exogenous salicylic acid (SA), sodium nitropusside (SNP, a nitric oxide donor), or their combination on dwarf polish wheat (Triticum polonicum L.) seedlings under UV-B stress were studied. The UV-B stress significantly decreased plant height, shoot dry mass, pigment content, net photosynthetic rate, intercellular CO₂ concentration, stomatal conductance, transpiration rate, and variable to maximum chlorophyll fluorescence ratio (F_v/F_m) in all plants, but less in the presence of SA, SNP, and their combination. On the other hand, there were considerable increases in malondialdehyde (MDA), proline, O₂ ^*-, and H₂O₂ content under the UV-B stress. When SA, SNP, and their combination were applied, content of MDA, proline, H₂O₂, and O₂ ^*- were less increased. Moreover, there were considerable increases in activities of superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione reductase under the UV-B stress and more in the presence of SA, SNP, and their combination. Therefore, it is considered that SA, SNP, and especially their combination could alleviate UV-B stress in dwarf polish wheat.

Polyploidization is a major genome modification that results in plant species with multiple chromosome sets. Parental genome adjustment to co-habit a new nuclear environment results in additional innovation outcomes. We intended to assess genomic changes in polyploid model species with small genomes using inter retrotransposons amplified polymorphism (IRAP) and retrotransposon microsatellite amplified polymorphism (REMAP). Comparative analysis among diploid and autotetraploid A. thaliana and A. suecica lines with their parental lines revealed a marginal fraction of novel bands in both polyploids, and a vast loss of parental bands in allopolyploids. Sequence analysis of some remodelled bands shows that A. suecica parental band losses resulted mainly from sequence changes restricted to primer domains. Moreover, in A. suecica, both parental genomes presented rearrangement frequencies proportional to their sizes. Overall rates of genomic remodelling events detected in A. suecica were similar to those observed in species with a large genome supporting the role of retrotransposons and microsatellite sequences in the evolution of most allopolyploids.

Malate is accumulated in grape pericarp until the start of ripening and then it is dissimilated. One aim of this study was to determine if the potential contribution of stored malate to the substrate requirements of metabolism in ripening grape pericarp is dependent on the cultivar. Two Vitis vinifera L. cultivars which accumulated different amounts of malate and had ripening periods of a different length were compared. The potential contribution of stored malate over the whole period of ripening was around 20 % in the cv. Sagrantino and 29 % in the cv. Pinot Noir. The contribution was higher in Pinot Noir because it contained more malate and had a shorter ripening period. A second aim of this study was to evaluate the contribution of gluconeogenesis to the amount of sugar accumulated in the pericarp. If all the dissimilated malate was utilized by gluconeogenesis, then the maximum contribution of stored malate to the total amount of sugar accumulated in the pericarp over the whole period of ripening was around 2.4 % in Sagrantino and 2.9 % in Pinot Noir. However, the actual contribution was only about 0.1-0.6 % in both cultivars because the majority of stored malate was not utilized by gluconeogenesis. However, it is likely that the actual contribution is much lower. This suggests that the function of gluconeogenesis is not to support accumulation of sugars in the fruits, but probably it plays other roles.

Molecular hydrogen (H₂) could be a novel signal in phytohormone signaling pathways in response to biotic and abiotic stresses. Here, we employed two wild rice species (Oryza rufipogon Griff. and O. minuta J. Presl) to test this hypothesis using hydrogen-rich water (HW). The expression differences of phytohormone and hydrogenase genes between conventional rice (Oryza sativa L,) and wild rice were determined by real-time quantitative polymerase chain reaction, and the effects of HW on gene expression of wild rice were detected during three growth stages. Expression of hydrogenase genes, synthesis genes, and receptor genes of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) signalling pathways was higher in six wild rice types than in conventional rice. Hydrogen-rich water up-regulated expression of two hydrogenase genes, SA, JA, and ET receptor genes and synthesis genes in the seedling stage of wild rice. But this positive regulation by HW was less significant in the vegetative and reproductive stages.

Ascorbic acid (AsA) is naturally occurring compound with antioxidant activity and plays a pivotal role in plant cell adaptation to salinity stress. The objective of this work was to assess the influence of exogenous AsA on the embryogenic callus of indica rice (Oryza sativa L.) cv. MRQ74 cultivated under saline conditions. NaCl (200 mM) decreased callus fresh and dry masses, relative growth rate, and K⁺ and Ca⁺² content, and increased Na⁺ content and Na⁺/K⁺ ratio. Application of AsA (0.5 or 1 mM) alleviated these effects of salinity. Activities of peroxidase, catalase, superoxide dismutase, as well as content of proline increased due to the NaCl treatment, and these parameters were mostly further increased by 0.5 mM AsA. Thus, AsA can increase callus tolerance to NaCl stress.

The Wx gene, which encodes waxy proteins, is the sole gene responsible for amylose synthesis in the wheat seed endosperm. In this study, we characterized, at the molecular level, several Wx alleles in durum wheat (Triticum turgidum L. ssp. durum Desf.) that had previously been catalogued at the protein level. Our data show a misclassification of the alleles in both Wx genes: Wx-A1 alleles from Blanqueta (Wx-A1a) and Astrodur (Wx-A1b) have been reclassified as Wx-A1a and Wx-A1h, respectively. A sequence comparison of the Wx-B1e allele shows that there were up to five different alleles under this denomination, which confirms that the protein analysis by SDS-PAGE separation needs to be combined with the PCR amplification-sequencing analysis in order to obtain a correct classification of the alleles, which will facilitate their use in wheat quality improvement.

Physiological changes associated with senescence of flowers and abscission of floral parts in Oncidesa (formerly Oncidium) cv. Gower Ramsey are caused by a plant hormone ethylene which is produced by pollinia cap dislodgment during postharvest handling and transportation. The ethylene receptor gene OgERS1 of Oncidesa has been previously cloned and characterized. To analyze promoter activity of OgERS1, transgenic Arabidopsis thaliana plants were generated to express the ß-glucuronidase (GUS) reporter gene under the control of 5'-upstream sequence of OgERS1 from Oncidesa. The expression pattern of the OgERS1 promoter at the cellular level was investigated by analysis of GUS activity. This promoter can activate gene expression in both actively dividing young tissues and abscission-related aging tissues. Expression of GUS was detected in the shoot meristem uniquely in 10 to 30 d-old-plants and was found in flower buds, axillary buds, flower stems, and abscission layers during later development. In 2- to 3-week-old transgenic Arabidopsis, exogenous ethylene, glucose, lactose, and maltose enhanced promoter activity implying that crosstalk between sugar and an ethylene receptor may exist. However, indole-3-acetic acid, benzylaminopurine, abscisic acid, heat, wounding, salinity, drought, and flooding slightly suppressed promoter activity. These results demonstrate that the promoter of OgERS1 was developmentally and environmentally regulated, and imply a potential for application of this bi-functional promoter to increase branching or enhanced dwarfing.

The nuclear factor Y (NF-Y) is one of the largest transcription factor families in plants consisting of NF-YA, NF-YB, and NF-YC subunits. It could play important roles in various processes such as flowering time, seed development, and response to drought. In this study, 6 NF-YA, 13 NF-YB, and 8 NF-YC proteins were identified and characterized in Prunus mume. Analyses of a conserved domain indicated that the PmNF-Y subunits shared an elevated degree of homology with the corresponding Arabidopsis NF-Y ones. Phylogenetic analysis showed that each NF-Y subunit family from Prunus mume and Arabidopsis could be divided into 4 or 2 clades based on their full-length proteins. The gene expression patterns of all 27 PmNF-Y genes were examined under abscisic acid (ABA), osmotic, salt, and H₂O₂ treatments using real-time quantitative PCR analyses. PmNF-YA1/2/4/5/6, PmNF-YB3/4/8/10/11/13, and PmNF-YC1/2/4/5/6/8 were found to be up-regulated under the ABA and osmotic treatments. PmNF-YA1/2/3/4/5/6, PmNF-YB1/3/8/10/11/13, and PmNF-YC1/2/5/6/8 were obviously induced by the H₂O₂. In addition, only PmNF-YA2 and PmNF-YB3 expressions were enhanced under the salt stress. These findings could provide an entry point to investigating the roles of PmNF-Y genes during abiotic stress responses.

Secretion of organic acids (OAs) by roots has been suggested to be an important mechanism of Al resistance in many species. In Stylosanthes, the participation of OAs in the mechanism of Al resistance is poorly understood. We aimed to study the production and secretion of OAs by two Brazilian Stylosanthes species with different Al resistance. Stylosanthes capitata and S. guianensis were treated with Al at different concentrations in 0.5 mM CaCl₂ (pH 4.0), and then root elongation, Al and OA content, OA secretion into the external solution, and the activity of citrate synthase (CS) were measured. Al-induced secretion of citric acid was also evaluated in the presence of protein synthesis and anion channel inhibitors. S. guianensis accumulated lower amounts of Al in its roots and displayed less inhibition of root elongation compared to S. capitata. Citric and malic acids were the most abundant OAs in the roots, and their content decreased with the Al treatment, except for citric acid in S. guianensis. Citric acid was the only OA secreted into the nutrient solution by the Al-treated plants of both species, but more by S. guianensis. Citrase synthase activity decreased in S. capitata but increased in S. guianensis with the Al treatment, and it may have a crucial role in the maintenance of citric acid content in the roots of S. guianensis. The use of anion channel and protein synthesis inhibitors reveal that anion channels were likely involved in the secretion of citric acid, and channel protein transcription was up-regulated by exposure to Al in Stylosanthes.

This study aimed to investigate sucrose-metabolizing enzymes and their genes in fruits of two longan (Dimocarpus longan Lour) cultivars Cihezhong (CHZ) and Lidongben (LDB). Content of sucrose, glucose, and fructose were measured by high-performance liquid chromatography. The genes of sucrose-metabolizing enzymes were cloned by combining reverse transcription polymerase chain reaction and rapid amplification of cDNA ends, and enzyme activities were analyzed at various points in the fruiting cycle. The total soluble solid (TSS) content of longan arils rose and was positively correlated with sucrose content during maturation and then declined as the fruit senesced. Cihezhong showed a more rapid decrease in sucrose content than LDB. The activities of both sucrose phosphate synthase (SPS) and sucrose synthase (SS) were lower in CHZ, whereas the activities of soluble acid invertase (SAI) and neutral invertase (NI) were higher. The full-length cDNA of the genes of the sucrose-metabolizing enzymes were cloned successfully. The patterns of changes of sucrose synthase-2 (DlSS-2), sucrose synthase-3 (DlSS-3), and neutral invertase-3 (DlNI-3) gene expressions corresponded to those of SS and NI activities. The rate of sucrose decline in the longan fruits was related to sugar receding, sucrose metabolizing enzyme activities, and corresponding gene expressions.

Germin and germin-like proteins (GLPs) are a broad and diverse family of developmentally regulated proteins widely distributed in plants. Oryza sativa L. harbours a large family of GLPs and serves as a good model for their study. In the present study, a germin-like protein gene (OsRGLP1) of rice origin was characterized by its heterologous expression in tobacco. The real-time PCR established almost a uniform expression of OsRGLP1 in leaves, stem, and roots of T₁ Nicotiana tabacum cv. Samsun. Although no morphological difference was apparent between T₀ transgenic and wild-type plants, leaves of mature transgenic plants showed necrotic lesions associated with an elevated content of H₂O₂, which was evidenced by in situ 3,3'-diaminobenzidine staining. A significantly higher activity of heat resistant superoxide dismutase (SOD) was observed in the transgenic plants as compared to the wild-type. The SOD activity in the transgenic plants was insensitive to potassium cyanide and sensitive to H₂O₂.

Phospholipase D (PLD) and its product phosphatidic acid are now considered to be one of the key elements of numerous physiological processes in plants including the salicylic acid signalling pathway. The presented study investigates the transcriptional regulation of Brassica napus PLDs following treatments with defense-related stimuli. We cloned eight B. napus genes encoding members of PLDβ, γ, and δ isoforms and performed phylogenetic analysis with its ancestor species Brassica rapa and Brassica oleracea, and with the model plant Arabidopsis thaliana. Transcription of the identified genes was monitored after treatment with benzothiadiazole (BTH), methyl jasmonate (MeJA), bacterial elicitor flg22, wounding, and after infection with fungal pathogens Sclerotinia sclerotiorum and Leptosphaeria maculans. Most of the genes responded specifically to a particular treatment. Remarkably the genes encoding the PLDγ and PLDβ isoforms were up-regulated by stimuli associated with the salicylic acid signalling pathway. The generality of this finding was confirmed by the analysis of public transcriptional data from Arabidopsis thaliana.

Eragrostis curvula (Schrad) Nees (weeping lovegrass) represents important cultivated forage in semiarid regions, and the most useful cultivars are tetraploid and reproduce by pseudogamous diplosporous apomixis. We previously produced a series of genetically related E. curvula lines that provide a suitable system for the identification of gene(s) involved in diplosporous apomixis and ploidy, including a natural apomictic tetraploid (T), a diploid sexual line (D), and a tetraploid sexual plant (C). A collection of expressed sequence tags (ESTs) was generated from cDNA libraries obtained from panicles of the D, T, and C, and leaves of the T. The present study aimed to analyze the repetitive content of these four cDNA libraries and further identify and characterize transposable element (TE)-related ESTs. Repetitive sequences were identified through the interface RepeatMasker (RM) using the database Repbase Update and further classification of TEs was performed manually from the RM output. The different contribution of ESTs with identity to TEs among libraries was further evaluated, and such differences were validated through RT-qPCR. We found that the percentage of repetitive content in the leaf cDNA library was almost double than in inflorescence libraries, with retrotransposons contributing mostly in all libraries. The expression of TE-related ESTs was compared in cDNA samples extracted from D, T, and C leaves or inflorescences revealing that seven mRNAs containing MuDR-like DNA transposons, Gypsy-like, and Copia-like retrotransposons were differentially represented according to tissue, reproductive mode, or ploidy. The euploid series of Eragrostis curvula is a useful model to the study of epigenomic changes produced after changes in ploidy. The present work constitutes the first detailed report on repetitive sequences of Eragrostis curvula at the transcriptome level.

In vitro orchid micropropagation is efficient biotechnological strategy for conservation and commercial plantlet production. However, micropropagated plantlets generally need to adapt to survive severe changes in humidity, irradiance, and growing medium that accompany the transfer to ex vitro conditions. Such adaptive cellular changes would give insights into the phenotypic plasticity of the model plant Cattleya xanthina (L.) Van den Berg. Therefore, we aimed to evaluate structural changes in the leaves of C. xanthina cultivated in vitro and acclimatized ex vitro using qualitative and quantitative analyses. During acclimatization, we observed a higher accumulation of dry mass, a greater convexity of the outer surface of epidermal cells, an increased deposition of epicuticular waxes, a greater elongation of mesophyll parenchymatic cells, and finally, the presence of chloroplasts with organized thylakoids and well-developed grana. Stomatal density was not changed. Furthermore, a gradual acclimatization allows this species the best adaptation to a new environment.

Calcium ion (Ca²⁺) is essential secondary messenger in plant signaling networks. In this study, the effect of Ca²⁺ on oxidative damage caused by a high irradiance (HI) was investigated in the leaves of two cultivars of tall fescue (Arid3 and Houndog5). Pretreatment of the tall fescue leaves with a CaCl₂ solution significantly increased Ca²⁺ content and intrinsic HI tolerance due to a decreased ion leakage and content of malondialdehyde, hydrogen peroxide, and superoxide radicals. Moreover, the activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase increased in both the cultivars in the presence of Ca²⁺ under the HI stress. In contrast, treatments with a Ca²⁺ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) or a plasma membrane Ca²⁺ channel blocker LaCl₃ reversed these effects. On the other hand, a pronounced increase in nitric oxide synthase-like activity and NO release by exogenous Ca²⁺ treatment was observed in the tolerant Arid3 plants after exposure to the HI, whereas only a small increase was observed in more sensitive Houndog5. Moreover, the inhibition of NO production by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or N ^ω-nitro-L-arginine blocked the protective effect of exogenous Ca²⁺, whereas the inhibition of Ca²⁺ by EGTA or LaCl₃ had no influence on the protective effect of NO. The results indicate that NO might be involved in the Ca²⁺-induced activities of antioxidant enzymes further protecting against HI-induced oxidative damage. This protective mechanism was found to be more efficient in Arid3 than in Houndog5.

Protein synthesis is a ubiquitous and essential process in all organisms, including plants. It is primarily regulated at translation initiation stage which is mediated through a number of translation initiation factors (eIFs). It is now becoming more apparent that in addition to synthesis of proteins, eIFs also regulate various aspects of plant development and their interaction with environment. Translation initiation factors, such as eIF3, eIF4A, eIF4E, eIF4G, and eIF5A affect different processes during vegetative and reproductive growth like embryogenesis, xylogenesis, flowering, sporogenesis, pollen germination, etc. On the contrary, eIF1A, eIF2, eIF4, and eIF5A are associated with interaction of plants with different abiotic stresses, such as high temperature, salinity, oxidative stress, etc. Similarly, eIF4E and eIF4G have roles in interaction with many viruses. Therefore, the translation initiation factors are important candidates for improving plant performance and adaptation. A large number of genes encoding eIFs can functionally be validated and utilized through genetic engineering approaches for better adaptability and performance of plants by inhibiting/minimizing or increasing expression of desired eIF(s).

Effects of four amino acids, L-asparagine, L-cysteine, L-citrulline, and L-glutamine in different concentrations (0, 0.5, 1, and 2 mg dm^-3) combined with 2 mg dm^-3 indole-3-butyric acid, on in vitro rooting and biochemical constituents of cherry rootstocks CAB-6P (Prunus cerasus L.) and Gisela 6 (P. canescens × P. cerasus) were investigated. In CAB-6P, root number and root fresh mass (FM) were maximum at 0.5 mg dm^-3 cysteine. All amino acids reduced root length in CAB-6P and root number as well as root FM in Gisela 6. In Gisela 6, 0.5 mg dm^-3 asparagine or 2 mg dm^-3 glutamine reduced root length. In CAB-6P, 100 % rooting was achieved in the control and with 1 and 2 mg dm^-3 cysteine or 1 mg dm^-3 citrulline. In Gisela 6, the rooting percentage was maximum (76.92 %) with 0.5 mg dm^-3 asparagine. Callus FM in CAB-6P was the greatest at 1 mg dm^-3 and in Gisela 6 at 2 mg dm^-3 citrulline. Callusing was 100 % in the majority of treatments for CAB-6P and 92.31 % for Gisela 6 with 0.5 or 2 mg dm^-3 citrulline. Cysteine, citrulline, and glutamine diminished chlorophyll content in Gisela 6 whereas in CAB-6P all four amino acids hardly affected it. Carotenoid and porphyrin content in CAB-6P was decreased due to asparagine (0.5 or 1 mg dm^-3). Porphyrin content in CAB-6P was also reduced by adding 0.5 or 1 mg dm^-3 cysteine or 2 mg dm^-3 citrulline. In Gisela 6, all amino acids decreased carotenoid and porphyrin content. In CAB-6P, all treatments except 0.5 mg dm^-3 glutamine or 2 mg dm^-3 asparagine increased leaf sucrose content. In roots, both sucrose and proline content were increased only at 1 mg dm^-3 cysteine whereas in leaves only 0.5 mg dm^-3 asparagine caused a 3-fold increase in proline content. A decrease in root proline in CAB-6P was observed due to asparagine, citrulline, or glutamine. In Gisela 6, decreased leaf sucrose and proline content was recorded at 2 mg dm^-3 cysteine. All amino acids did not alter root sugar content remarkably whereas root proline content was raised by adding 0.5 mg dm^-3 glutamine or 1 mg dm^-3 cysteine.

To investigate the antioxidative response of glutathione metabolism in Urtica dioica L. to a cadmium induced oxidative stress, activities of glutathione reductase (GR), glutathione-S-transferase (GST), and glutathione peroxidase (GSH-Px), content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation (LPO), and also accumulation of Fe, Zn, Mn, Cu besides Cd were determined in the roots, stems, and leaves of plants exposed to 0 (control), 0.045, and 0.09 mM CdCl₂ for 58 h. Whereas the Cd content continuously increased in all organs, the Fe, Zn, Mn, and Cu content decreased in dependence on the applied Cd concentration and incubation time. The Cd treatment resulted in increased GR and GST activities in all organs, however, GSH-Px activity was dependent on Cd concentration and plant organ. The GSH/GSSG ratio maintained above the control level in the stems at both Cd concentrations. The LPO was generally close to the control values in the roots and stems but it increased in the leaves especially at 0.09 mM Cd.

Desiccation tolerance (DT) is the ability to tolerate dehydration to levels below 0.1 g(H₂O) g^-1(dry mass) and subsequent rehydration without lethal damage. Here, it is proposed that Leucaena leucocephala, a tree species, has potential to be model tolerant species in seed research. Using flow cytometry and transmission electron microscopy, cytological changes related to loss of DT in Leucaena primary roots were followed during germination. Leucaena seeds lost their DT at the end of germination and this coincided with an increase in cellular 4C DNA content. A negative correlation between the 8C DNA content and the capacity of germinating Leucaena seeds to tolerate desiccation was also observed. Apparently, the seeds of Leucaena underwent extra cycles of endoreduplication and accumulated a high content of DNA - an event not previously linked to DT. The ultrastructural damage imposed by drying overcame Leucaena primary root cell resilience and their ability to resume normal growth. Nuclear DNA content may be used as indicator of progress of germination and loss of DT in Leucaena.

Licorice (Glycyrrhiza glabra L.) is an important medicinal plant accumulating high-value secondary metabolites. Real-time reverse transcription quantitative PCR (RT-qPCR) has become a common method for studying gene expression, and the availability of stable reference genes is a prerequisite to obtain accurate quantification of transcript abundance. Therefore, an experiment was designed to determine appropriate reference genes for gene expression studies in licorice. Based on reports in the literature and the availability of genomic sequences, eight putative reference genes were chosen. Further, the expression stabilities of these genes were evaluated in leaf and root tissues under normal and drought stress conditions using three distinct statistical algorithms including geNorm, NormFinder, and BestKeeper. Among the investigated genes, ubiquitin-conjugating enzyme E2 (UBC2), elongation factor 1 α (EF1), and actin (ACT) under normal conditions and ACT, β-tubulin (BTU), and UBC2 under drought stress conditions were the most stable genes in leaves, whereas BTU, ACT, and UBC2 under normal and drought stress conditions were identified as the most stable genes in roots. Nevertheless, the use of glyceraldehyde-3-phosphate dehydrogenase, F-box protein, and BTU have not been approved as reference genes for RT-qPCR data normalization. The findings in this study highlight the importance of the use of well-validated reference genes to the success of gene expression analysis using RT-qPCR.