Strawberry is an economically important fruit crop worldwide. Circadian clock genes are endogenous timers that regulate a wide range of metabolic processes and consequently plant development. However, little is known about the circadian clock genes in strawberry. In the present work, we identified 12 primary circadian clock genes from the diploid woodland strawberry (Fragaria vesca L.) genome. Phylogenetic, conserved motif, and gene structure analyses revealed the evolutionary relationships of strawberry circadian clock genes with homologous genes from other species. Promoter analysis revealed different regulatory elements responding to abiotic and biotic stresses and phytohormones. We characterized the transcript patterns of strawberry circadian clock genes over a 48-h period. The expression patterns of seven circadian clock genes displayed circadian rhythms. We also examined the expression patterns of these genes in response to low-temperature stress and six of them showed an upregulated expression. Interestingly, most of these upregulated genes were highly expressed during the day. Our study reveals the characteristics of primary circadian clock components in diploid woodland strawberry and their responses to low-temperature stress and lays a foundation for future functional studies of these circadian clock genes during the growth and development of diploid woodland strawberry.

In plants, class III T2 RNases involves two groups of structurally similar proteins, but with different biological functions: S-RNases and non-S-RNases. S-RNases have been involved in self-incompatibility whereas non-S-RNases have been implicated in stress responses. Here we report a novel class III RNase termed NaPi/S_x-RNase, which works both in self-incompatibility and in response to phosphate deficiency. The NaPi/S_x-RNase gene was identified in roots of Nicotiana alata grown in the absence of inorganic phosphate. Phylogenetic analysis showed that NaPi/S_x-RNase was included within the class III RNase T2 group. The NaPi/S_x-RNase was expressed in styles and its temporal expression increased in parallel to stylar development, with a slight decrease after anthesis. Progeny analysis showed that NaPi/S_x-RNase and S₁₀₇-RNase, a functional allele of the self-incompatibility system, segregated in a 1:1 ratio. The progeny segregation of a semicompatible cross, in which NaPi/S_x-RNase was shared by the two parents, exhibited a pattern consistent with a functional S-RNase allele. Considering genetic segregation, primary structure, and physiological role, the NaPi/S_x-RNase may be either an S-RNase with diversified functions or a non-S-RNase linked to the S-locus. To our knowledge, this is the first evidence for a specific function of the S-locus other than the self-incompatibility reaction. These results support the hypothesis that the self-incompatibility and stress responses may have evolved from a common origin.

Elymus nutans is an important alpine perennial forage of the Pooideae subfamily, that can survive subzero temperatures. To understand the molecular mechanisms underlying cold tolerance in E. nutans, we performed the transcriptional analysis by RNA-Seq in two genotypes, the tolerant Damxung (DX) and the sensitive Gannan (GN), under cold stress. The new E. nutans transcriptomes comprised 200 520/200 836 and 181 331/211 973 transcripts in leaves/crowns of DX and GN, respectively. More cold-stress-related genes were identified in leaves than in crowns of both genotypes throughout the whole cold stress. The most prominent functional category in leaves of both genotypes at 3 h of stress was transcriptional regulation. Brassinosteroid and jasmonic acid mediated signalling pathways play central roles in regulating downstream protective responses in DX after 24 h of cold stress. Prolonged cold stress caused more severe transcriptome responses in crowns and leaves of DX compared to GN. The most significant transcriptomic changes in both genotypes were associated with the response to abiotic stresses and the oxidation-reduction processes, implying reprogramming of the cellular metabolism as an adaptation to cold stress. This study reveals mechanisms of genotype- and organ-specific cold stress response in E. nutans and thus provides a basis for future breeding strategies aimed at improving the tolerance of cold-sensitive plants.

Leek is an economically important vegetable. In model plants, the CONSTANS (CO) and CONSTANS-like (COL) genes play central roles in plant flowering modulation. However, none of leek CO homolog has been identified, because of limited gene resources obtained in this crop. Here, we reported the transcriptome analysis of leek, along with the identification of putative leek CONSTANS-like (COL) (ApCOL) genes. A total of 189 713 non-redundant transcripts were de novo assembled by using about 128.9 million clean sequence reads, of which, 48 621 were achieved for functional annotation. Thereafter, the search for putative ApCOL genes against the assembled transcripts was performed, and 17 genes were identified. The 17 putative ApCOL proteins, together with 16 function-known COL proteins published for other species, were subjected to phylogenetic analysis and divided into four groups. Some putative ApCOL members showed high sequence similarity with published COL proteins involved in flowering regulation. Expression analysis revealed that, among the 17 putative ApCOL genes, eight, two, and three genes showed higher expression in leaves, cauloids, and roots, respectively. The discovery of putative ApCOL genes and the characterization of their expression patterns will provide a basis for future clarification of their functions in leek growth and development.

Root hairs play important roles in plant nutrient and water acquisition. To better understand the genetic mechanism controlling root hair development in rice (Oryza sativa L.), a rice mutant with root hair defects was isolated and characterized. Cryo-scanning electron microscope (SEM) showed that the density and length of root hairs in the mutant were significantly reduced compared to wild type (WT). Map-based cloning and complementation test revealed that the mutation occurred in a NADPH oxidase gene OsNOX3 (LOC_Os01g61880). The OsNOX3 displays high sequence similarity with the previously characterized NOX genes RTH5 in maize and RHD2 in Arabidopsis, which play critical roles in root hair development. Expression pattern analysis indicated that OsNOX3 is expressed in various tissues throughout the plant with high expression in roots and root hairs. Subcellular localization analysis confirmed that OsNOX3 is located on the plasma membrane. Staining assays showed that the content of superoxide and hydrogen peroxide are significantly reduced in root hair tips of Osnox3 when compared to WT. Our results showed critical roles of OsNOX3 in regulating both root hair initiation and elongation in rice, which is similar to RTH5 but different from RHD2, confirming the difference of genetic mechanisms regulating root hair morphogenesis in monocot and dicot plants.

The MYB transcription factor superfamily is a large gene family that plays central roles in developmental processes and defence responses in plants. Unlike in Arabidopsis, only few members of the R2R3-MYB gene family have been functionally well characterized in maize, especially in abiotic stress-response pathways. Subgroup-specific conserved motifs outside the MYB domain may reflect functional conservation. A comparative genomics study using Arabidopsis abiotic stress-responsive MYB protein sequences identified 46 ZmMYB genes that may be involve in abiotic stress responses of Zea mays. An expression pattern analysis of the 46 ZmMYB genes under abiotic stress treatments was used to identify 22 MYB genes that were induced by one or more of the stress treatments. ZmMYB30 was highly upregulated under the four stress treatments. The ectopic expression of ZmMYB30 in transgenic Arabidopsis plants promoted salt-stress tolerance and also increased the expression of a number of abiotic stress-related genes, allowing the plants to overcome adverse conditions.

Glucosinolates are a branch of amino acid-derived metabolites, which are specifically found in Brassicales. In Arabidopsis, tryptophan derived indolic glucosinolates are required for plant defense against a wide range of pathogens and herbivores due to their strong antimicrobial activity and potential signaling function. An important enzyme in indolic glucosinolate biosynthesis pathway is CYP83B1, which oxidizes indole-3-acetaldoxime, a precursor of indole-3-acetic acid (IAA). In this study, we reported isolation and expression characterization of a CYP83B1 gene from Brassica oleracea L. var. italica Plenck, which we termed BoCYP83B1. Overexpression of BoCYP83B1 in Arabidopsis resulted in an altered glucosinolate profile and early flowering phenotype. By expressing the reporter gene β-glucuronidase under the control of the BoCYP83B1 promoter in Arabidopsis, we analyzed the spatial expression pattern of BoCYP83B1 under normal growth conditions as well as in response to several hormones and stresses. The BoCYP83B1 was primarily expressed in vascular tissue through the almost whole plant. It was strongly induced by methyl jasmonate, 1-amino-1-cyclopropanecarboxylic acid, salicylic acid (SA), gibberellin, and IAA, suggesting its involvement in complex signaling pathways. Mannitol, NaCl, UV, and Flagelin 22 significantly up-regulated BoCYP83B1 expression, indicating its possible role in stress response. Interestingly, the response of BoCYP83B1 to SA and NaCl showed tissue specificity. Thus, BoCYP83B1 might have different functions in different tissues.

Finger millet (Eleusine coracana) is one of important crops, and its grains contain an exceptionally high content of calcium. In order to investigate the molecular mechanism by which it orchestrate the accumulation of Ca²⁺ during grain filling, some candidate genes encoding calcium transporters [calcium exchangers (CAX1, CAX3)] and sensors [calcineurin-B like (CBL4 and 10)], a CBL-interacting protein kinase (CIPK24), and calmodulin (CaM) were identified using transcriptomics and differential expression analysis in two genotypes of finger millet differing in grain calcium content. These transporters and sensors are highly expressed in leaves and developing spikes of the genotype with a high grain Ca²⁺ indicating their potential role in Ca²⁺ accumulation. Calcium transporters, mainly CAXs, pump Ca²⁺ inside the cell through plasmalemma and tonoplast, and their activities are regulated by CaM dependent and independent Ca²⁺ sensor proteins of CaM and CBL-CIPK networks. Abundance of CaM in a high grain Ca²⁺ genotype is suggestive that CaM might also contribute for grain calcium accumulation by interaction with Ca²⁺ATPase. The upregulation of CAX1 in vegetative tissues and developing spikes and CAX3 only in developing spikes provides the most plausible clue for calcium transport and accumulation regulated by tripartite interaction in finger millet.

Medicinal plants are a rich source of secondary metabolites, extensively used in traditional health care systems. High altitude biodiversity encompasses the diversified and valuable medicinal plant species. The extreme environmental conditions of high altitude region viz. fluctuating temperatures, high UV radiation, salinity, low oxygen concentration, and high wind velocity limits the plant growth and distribution. Yet, how medicinal plants respond to these extreme conditions is not sufficiently understood. Therefore, addressing plant acclimation to different stresses presents an opportunity to unravel adaptive mechanism of medicinal plants along altitude gradient. This article reviews the recently published research that highlights the major role of proteins in plant adaptation to extreme environmental conditions. In the last few decades, climate change has made a profound impact on high altitude plants. Stress conditions alter cellular homeostasis of plants. With the advent of proteomics, it has become evident that stresses induce changes in proteome by synthesis/expression of novel stress responsive proteins. These proteins constitute a highly organized, complex network that leads to changes in the molecular, biochemical, physiological, and morphological responses of plants. Herein, we comprehensively discuss the proteomics of medicinal plants and its role in adaptation along altitude gradient. This review aims to provide impetus to current research in medicinal plants ranging from developmental to stress biology and to generate basis for genetic engineers and plant breeders to produce next-generation medicinal plants.

A large-scale expression profiling study was performed to investigate candidate genes associated with the two quantitative trait loci (QTLs) for aluminum (Al) tolerance (Alt1 and Alt2). They have been identified in rye and localized on chromosomes 6R and 3R, respectively. Materials employed were hexaploid wheat (cv. Chinese Spring), and two wheat-rye addition lines (3R-AL and 6R-AL). Seedlings were treated with and without Al for 24 h to examine genes up-regulated or down-regulated by Al. Measurements of root growth at different Al concentrations showed the Al tolerance was higher in 3R-AL than in 6R-AL and wheat. Initial transcriptomic results revealed that more genes changed expression (>10 fold) in the wheat and in the 6R-AL line (moderately tolerant) than in the 3R-AL line (highly tolerant). A method was developed to determine whether candidate genes are involved in Al tolerance or in responses to Al toxicity. Real-time qPCRs were carried out in a subset of six genes with known function in near isogenic rye lines 389 (Al-sensitive) and 390 (Al tolerant). All six genes were up-regulated by Al in line 389 but not in line 390, indicating that they were involved in Al stress response but not in Al tolerance mechanisms. Subsequent analysis of Arabidopsis lines with knockout mutations in homologues of these six genes showed an Al sensitivity similar to the wild-type, providing more evidence towards their participation in the response to stress rather than to Al tolerance. Once the stress response genes were ruled out, the focus was turned to the identification of tolerance genes by studying transcripts up-regulated and down-regulated in the tolerant 3R line with respect to wheat and 6R line. Finally, a list of candidate genes that could be conferring increased tolerance was obtained.

Hemp (Cannabis sativa L.) seeds have been recognized as a nutritional protein source for humans and animals. In this study, gene families encoding precursor polypeptides of three storage protein classes, including six 11S edestin, two 2S albumin and one 7S vicilin-like genes were identified and characterized from an inbred line of hemp. All edestins showed typical 11S globulin features but based on the amino acid composition, they were grouped in three edestin types (type1, -2 and -3). Genes encoding edestin type1 and -3, were very close to each other in a DNA fragment of 16 071 bp, whereas the two isoforms of edestin type2 were linked on a different DNA fragment of 8 232 bp and arranged in a tailto- tail fashion. All edestin types were very rich in arginine and glutamic acid, but edestin type3 was the richest in cysteine and methionine. Regarding the 2S albumin (Cs2S) two genes were identified in a fragment of 13 738 bp in a tail-to-head array. Finally, only one 7S-vicilin like gene (Cs7S) that exhibited typical 7S vicilin features such as the presence of two cupin domains and several N-glycosylation sites was isolated. Southern blot hybridization is in agreement with the number of genes isolated, and real-time qPCR analysis revealed that all genes are expressed in the seed. The highest expression was observed for edestin type1 (CsEde1) and Cs2S, whereas the lowest expression was detected for Cs7S. The results of this study provide a complete overview of the genes encoding hemp storage proteins and significantly advance our knowledge on the organization of these gene families.

Rapid and non-destructive assessment of water status is essential to enhance crop performance. This study aimed to evaluate photosynthetic performance and to monitor water status in cotton under field conditions. A two-year experiment was conducted with three irrigation regimes to measure the following parameters: photochemical reflectance index (PRI), structural independent pigment index (SIPI), water index (WI), solar-induced fluorescence (SIF; retrieved from reflectance by using Fraunhofer line-depth method), gas exchange, and chlorophyll fluorescence (CF). The results showed that PRI decreased > 30 % in moderate drought (MD) and >50 % in severe drought (SD), compared with control. PRI was found to be positively correlated with net photosynthetic rate (P_N), stomatal conductance (g_s), transpiration rate (T_r), actual quantum yield of photosystem II photochemistry (Φ_PSII), but a negatively correlated with nonphotochemical quenching (NPQ). Solar-induced fluorescence around 761 nm (SIF₇₆₁) had significant correlations with P_N, Φ_PSII, and NPQ, but not with maximal quantum yield of PS II photochemistry (F_v/F_m). The relationship between PRI and PN was stronger at the beginning of water stress (R² = 0.86) than for the all stress stages (R² = 0.54), indicating that PRI could be more effective for assessing PN of cotton at early water stress. PRI was better correlated with relative water content and photosynthetic parameters than SIPI and WI and so it could also be a good indicator to evaluate cotton water status.

Low temperature can affect the growth and development of lily, limiting the application of commercial cultivars in outdoor. Lilium lancifolium is an important cold-resistant wild lily, but little is known about how L. lancifolium tolerates cold stress at the molecular level. In this study, we identified and characterized genes and transcription factors associated with cold stress in control plants and plants treated by 4° C for 1 - 24 h. The construction of a highest reciprocal rank-based gene co-expression network along with its partition into defined functional modules using Markov cluster algorithm resulted in identification of 30 gene modules and some of them were significantly enriched with various kinds of stress response under 4° C. These gene modules were associated with metabolic processes, cellular processes, regulation of biological processes, establishment of localization, and responses to stimuli. Moreover, three transcription factors that may regulate the downstream genes involved in response to stimuli were also found. We further studied the expression pattern and tissue specificity of these transcription factors. The functional evaluation of the various interesting genes in this study will probably provide novel discovery of pathway members and regulators associated with cold resistance in lily.

Candidate gene association studies implicate the detection of contributing single nucleotide polymorphism (SNP) for the target traits and have been recommended as a promising technique to anatomize the complex characters in plants. ERECTA gene in plants controls different physiological functions. In this study, we identified SNPs in 1.1 kb partial sequences of TaER-1 and TaER-2 of wheat (Triticum aestivum L.). Thirty-nine SNPs were identified in the coding regions of TaER-1 gene in 33 wheat genotypes, of which 20 SNPs caused non-synonymous mutations while 19 SNPs produced synonymous mutations; while 31 SNPs were located in the coding regions of TaER-2 gene in 26 genotypes, of which 18 SNPs caused non-synonymous mutations and 13 SNPs caused synonymous mutations. In addition, 32 SNPs in TaER-1 and 9 SNPs in TaER-2 were also identified in the non-coding regions. Moreover, the significant genetic associations of SNPs of TaER-1 and TaER-2 genes with carbon isotope discrimination, stomatal conductance, photosynthetic rate, transpiration rate, intrinsic water use efficiency (iWUE), leaf length, leaf width, stomatal density, epidermal cell density, and stomatal index were noted in wheat genotypes. This study confirms the importance of TaER-1 and TaER-2 genes which could improve iWUE of wheat by regulating leaf gas exchange and leaf structural traits. These identified SNPs may play a critical role in molecular breeding by means of marker-assisted selection.

Flood in rice fields at the time of seed sowing and early seedling establishment causes extensive crop loss due to the inability of the seeds to tolerate and overcome submergence. In the present study, rice genotypes from Assam, India tolerant to hypoxia during germination were identified through systematic screening of deep-water rice genotype collection from flood-prone ecosystem of Brahmaputra river valley. The difference in tolerance to hypoxia during germination within the species provides scope for identification of novel hypoxia-responsive factors involved in tolerance as mechanism of tolerance may not be conserved across tolerant germplasms. Tolerant genotypes were further subjected to physiological and molecular characterization. Growth rate kinetics in tolerant genotypes new Rangdhakekua bao (RKB) and cultivar Khao Hlan On (KHO) from International Rice Research Institute (IRRI) used as positive control exhibited stronger escape strategy under hypoxic condition compared to sensitive genotype IR-64 (negative control). Activities of α-amylase and pyruvate decarboxylase were significantly higher in RKB and KHO than in IR-64, while no significant difference was observed in the alcohol dehydrogenase activity. Reverse transcription quantitative PCR confirmed increased amounts of transcripts of sucrose nonfermenting 1 related protein kinase, myeloblastosis-related protein S1, rice amylase 3D, and trehalose phosphate phosphatase 7 genes, which are known to be involved in hypoxia signaling cascade. Besides, transcription factors (TFs) like ethylene responce factors 71 and 63, ethylene insensitive 3-like 1a and proteins like expansins A7 and A2, which are involved in cell elongation had also significantly higher amounts in RKB compared to IR-64. Additional factors that include TFs like ERF71 and ERF63 that shows perturbation at transcription even within tolerant genotypes might constitute the genotype-specific regulation, evolved as a part of its adaptive mechanism to survive under submerged conditions.

The aim of this study was to evaluate the tensile strength of the rachilla in spikelets of Polish cultivars of Festulolium braunii (K. Rich.) A. Camus during seed development and maturation. The investigations were carried out in 2009 - 2010 at the Plant Breeding, Szelejewo, Poland. The inflorescences were randomly harvested in June - July from plant collection in maintenance breeding of three Polish cultivars (Agula, Felopa, and Sulino). Using a specifically developed testing machine, tensile strength of the rachilla of individual spikelets were determined. Regardless of the F. braunii cultivar, the tensile strength of the rachilla decreased through consecutive growth stages. The highest tensile strength of the rachilla was observed during the phase of kernel watery ripe stage (BBCH 71) - it ranged from 510.1 mN in 'Agula' to 592.0 mN in 'Felopa'. At the fully ripe phase (BBCH 89), the value of this trait ranged from 19.2 mN in 'Agula' to 45.0 mN in 'Felopa'. It was also observed that in all tested cultivars of F. braunii, the spikelets located in the lower part of the spike were characterised by the highest tensile strength of the rachilla, whereas those in the upper part were characterised by the lowest values of this trait. This means that the caryopses in the spikelets located at the lower part of the inflorescence were less susceptible to shedding. Seed shedding the Polish F. braunii cultivars, especially 'Agula' and 'Sulino', may start as early dough and soft dough phases. By contrast, seed shedding 'Felopa' is moved to the later phases of seed maturation.

The reverse transcription quantitative real-time PCR (RT-qPCR) is becoming increasingly important for gene expression studies. However, the accuracy and reliability of RT-qPCR depend on normalizing expression to reference genes. In this study, ten candidate reference genes, including cyclophilin (CYP), elongation factor 1b (EF1b), α-tubulin (TUA5), β-tubulin (TUB4), ubiquitin10R (UBQ10R), 60S ribosomal RNA (60S), alcohol dehydrogenase (ADH3), metalloprotease (MTP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT2) were evaluated for the stability of expression in three tissues of two peanut cultivars [Zhonghua 2(ZH₂) and 99-1507] under Al stress by four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder). The results suggested that the top-ranked reference genes under Al-induced programmed cell death (PCD) in peanut were UBQ10R, EF1b and CYP, with the most suitable combination of reference genes being [UBQ10R+ACT2]. The UBQ10R exhibited the most stable expression in all samples, while TUB4 was the least stable gene. The relative expression of AhMC1 (the caspase-like protease family gene, which played a significant role in Al-induced PCD) showed that there was no significant difference with the best reference gene and the best gene combination in RT-qPCR normalization, but there was significant difference with the least stable gene TUB4 as reference gene. This is the first study to evaluate the stability of reference genes in peanut under Al-induced PCD, and the results will provide guidance to identify appropriate reference genes for further RT-qPCR analyses under Al stress in peanut.

Melatonin (N-acetyl-5-methoxytryptamine) is an essential molecule which regulates plant growth and development and alleviates the damaging effects of abiotic stresses. To evaluate the important functions of melatonin in response to salinity stress, the effects of exogenous melatonin on the antioxidant system and growth of tomato (Solanum lycopersicum L.) under 150 mM NaCl stress were investigated. The application of 100 μM melatonin compensated the growth inhibition caused by salt-stress. Melatonin treated seedlings had an increased fresh and dry masses of shoots and roots. The application of 1 - 200 µM melatonin notably enhanced the relative chlorophyll content (SPAD index), root characteristics, and gas exchange in tomato seedlings subjected to salt stress compared to seedlings treated with salt stress alone. Moreover, melatonin pretreatment minimized accumulation of reactive oxygen species and improved activities of antioxidative enzymes including catalase, superoxide dismutase, glutathione reductase, and ascorbate peroxidase.

Cucumber (Cucumis sativus L.) is considered a model plant for the investigation of Fe deficiency responses, since it strongly exhibits typical strategy I, i.e. increased activities of Fe(III)-chelate reductase, H⁺-ATPase and iron regulated transporters. In this study, cDNA amplified fragment length polymorphism analysis was employed to identify genes differentially expressed in the root apex following Fe deficiency. The expression patterns of the most interesting transcript derived fragments were validated by semiquantitative reverse trascriptase - polymerase chain reaction. A set of new genes overexpressed under Fe deficiency, such as those coding for calmodulin, SNAP, TIM23 and V-PPase were identified. Furthermore, we also observed that calmodulin protein accumulated in Fe-deficient root apexes.

Circular RNAs (circRNAs), an emerging class of non-coding RNAs, are abundant in eukaryotic transcriptomes. Seed germination is one of the most important stages in the entire life cycle of plants that can be slowed down or totally restrained by high temperature. Our aim is to identify heat-responsive circRNAs and explore the potential function of circRNAs in tomato seeds at high temperature. Following high-throughput sequencing, 4 164 circRNAs were identified, and 980 circRNAs were shared in the control and high-temperature libraries. Among the 748 circRNAs with high expressions, 73 circRNAs were significantly up-/down- regulated in tomato seeds germinated at high temperature compared to the control. The parental genes of circRNAs existing in seeds only at high temperature were mainly involved in metabolic processes, cellular processes, catalytic activities, and binding based on Gene Ontology analysis. The results suggested that circRNAs were widespread in tomato and were generated from different chromosomes and diverse genomic regions. Some circRNAs in tomato seeds responded to high temperature during germination. This study provides the first genome-wide profile of circRNAs in response to high temperature during tomato seed germination and lays a foundation for studying the potential biological functions of circRNAs responding to heat stress.

In order to introduce drought tolerance and improved cell wall digestibility from fescue in fodder ryegrasses, we developed two amphiploid Festulolium synthetics. One is a synthetic composed of three selected drought tolerant F1 hybrid genotypes from a cross between tetraploid Lolium multiflorum and hexaploid Festuca arundinacea, further on called LMFA. The other is a synthetic composed of five selected genotypes with soft leaves from a cross between tetraploid Lolium perenne and tetraploid Festuca pratensis, further on called LPFP. We produced seeds in polycrosses of two generations of both amphiploids, i.e., syn1 and syn2, and tested them in plot trials to determine the yield and fodder quality. The syn1 of both Festulolium populations had a higher annual dry matter yield than the reference Lolium cultivars and Festulolium cultivars composed of the same parental species. However, the syn2 of LMFA did not show an improved drought tolerance during a dry growing season compared to other Festulolium cultivars, and the seed yield of LMFA syn1 was low and dropped extremely in syn2. The number of chromosomes of LMFA also decreased gradually from F1 to syn2, and there was a clear shift in chromosome composition towards the Lolium genome. The LPFP synthetic performed better. Although the sugar content was significantly lower than the sugar content of the perennial ryegrass cultivars, organic matter digestibility (OMD) of LPFP was as high as OMD of the tetraploid perennial ryegrass cultivars. The cell wall digestibility (NDFD) of LPFP was significantly higher than the NDFD of both parental species and higher than the NDFD of all tested Festulolium cultivars. The seed yield of LPFP was the same in syn1 and syn2. The chromosome number remained on average the same and no clear shift of the chromosome composition to one of the composing genomes was observed. Overall, chromosome analysis revealed a high number of aneuploidy in syn1 and syn2 generations of both LMFA and LPFP and a lot of variation in number of Lolium, Festuca and recombinant chromosomes, and in the Lolium:Festuca genome ratio was observed among different genotypes of the same population. Therefore, selection for genotypes with a more stable genome composition will be a prerequisite for a sufficient seed yield and a broader exploitation of these new Festulolium synthetics.

Heat stress is one of the most crucial factors affecting crop growth and productivity worldwide. So, searching for a potent eco-friendly heat stress alleviator is the main issue nowadays. The current study was conducted to assess the ameliorative effects of 1.5 mM potassium silicate (K₂SiO₃, further only Si) or 1.66 mM silicon dioxide nanoparticles (SiNPs) on wheat (Triticum aestivum L.) seedlings exposed to heat stress (45 °C, 4 h). The observations show that Si or SiNPs treatments significantly restored the heat stress-provoked ultrastructural distortions of cellular organelles, particularly chloroplasts and the nucleus. Further, both Si and SiNPs enhanced the photosynthetic capacity as revealed by increments in the photochemical efficiency of photosystem II and the performance index as well as the content of photosynthetic pigments. A reduction in malondialdehyde accumulation in Si and SiNPs treated plants was positively related to their membrane stability index. The reverse transcription PCR analysis showed that Si treatment but not SiNP treatment stimulated the overexpressions of both Triticum aestivum plasma membrane intrinsic protein (TaPIP1) and Triticum aestivum nodulin 26-like intrinsic protein (TaNIP2) aquaporin genes parallelly with an improvement in the relative water content. This investigation reveals that Si was more effective than SiNPs in restoring the heat stress injuries. To the best of our knowledge, this is the first investigation exploring the effects of Si and SiNPs in improving thermotolerance of wheat seedlings.

Bamboo possesses many unique physiological characteristics, but the molecular understanding of many of these processes remains poorly understood till to date. One major reason is unavailability of sufficient sequence and expression data. Selection of suitable reference genes is pivotal to initiate any gene expression analyses. Although, suitable reference genes have been identified in the temperate bamboo Phyllostachys edulis, it has not been done for tropical bamboo. In this study, expression stability of 10 candidate reference genes were investigated in 4 widely grown tropical bamboo species (Bambusa tulda, B. balcooa, B. bambos, and B. vulgaris), different organs (young leaves from flowering and non flowering culms, flag leaf (leaf just below the mature inflorescence), possible flag leaf (leaf covering the immature inflorescence), culm sheath, internode, root, rhizome, and inflorescence bud), different parts (basal, middle, and tip regions of leaf; internodes located in the basal, middle, and tip region of the branch, and developmental stages early, middle, and late inflorescence buds) by using 3 reliable computational tools (geNorm, NormFinder, and RefFinder). A universal single reference gene for normalization of gene expression data was not identified. However, the eukaryotic initiation factor 4α (eIF4α), clathirin adaptor complexes medium subunit (CAC), and nucleotide tract-binding protein (NTB) were found stable in the selected organs across different bamboo species. On the other hand, eIF4α ranked top when different organs and peptidyl prolyl cis-trans isomerase/cyclophilin (CYP), eukaryotic elongation factor 1α (eEF1α) and ubiquitin 5 (UBQ5) ranked top when different developmental stages of B. tulda were analyzed. Taken together, this study not only identifies reference gene/s that are stable across species, organs, and developmental stages of bamboo, but it also assesses the impacts of major contributing factors regulating expression stability of the reference genes.

Stomatal guard cells are specialized epidermal cells regulating gas exchange. The ability to open or close in response to external and internal cues makes stomata a dynamic and fascinating system. Stomatal closure upon infection ensures restriction of pathogen entry into the plant and forms an essential component of innate immunity. The opening or closure of stomata is dependent on the turgidity or flaccidity of guard cells, respectively, facilitated by several signaling components, including reactive oxygen species, nitric oxide (NO) and Ca²⁺. Among these, NO is the most extensively studied gasotransmitter. Its pivotal role in stomatal closure by modulating various downstream components as well as regulation of crucial proteins by post-translational modifications makes NO an essential factor. Two more gasotransmitters, carbon monoxide and hydrogen sulfide, also trigger stomatal closure. Other gaseous molecules, like ethylene, methane, sulfur dioxide, ozone, and CO₂, can modulate stomatal closure, but they are not considered strictly as gasotransmitters due to specific criteria. We review the signaling events in guard cells triggered by these gasotransmitters leading to stomatal closure. We point out the dual role of NO to promote stomatal closure and stomatal opening. Both NO and H₂S help in reinforcing the innate immunity against pathogen attack. Although there is extensive information on the mechanism of NO action on stomata, the enzymatic source of NO or CO is still ambiguous. Similarly, research is warranted to establish the relative importance of and interactions among the three main gasotransmitters. Further studies on gasotransmitters would answer the ambiguity about their functions and confirm if they can act independently.

Small RNAs (sRNAs) are essential components of gene-regulatory networks, which guide plant development and tune it to environmental challenges. Though the past years have witnessed evidences on sRNA importance for stress response, there is scarce data on their involvement in resurrection plant survival under severe drought. Haberlea rhodopensis (hrh) is an angiosperm resurrection species, whose vegetative tissues can tolerate desiccation and recover upon rehydration. In this study, high-throughput sequencing sRNAs indicated a higher complexity of the sRNA population, especially of a 24 nt sRNA category, in the desiccated vegetative tissue of H. rhodopensis compared to unstressed tissues. The cross-species discovery was performed to predict 77 mature microRNAs (miRNAs), most of which were assigned to 23 high-confidence conserved miRNA families in the leaf tissue. Several members of the miR156/157, miR166, and miR399 families were found to be desiccation-responsive. The miR156/157 family members were found up-regulated upon dehydration and down-regulated upon rehydration, while the miR166 and miR399 family members followed an opposite trend of expression. A probable miR156/157 target, orthologous to the SQUAMOSA promoter binding protein-like, was reconstructed in H. rhodopensis based on genomic data available for this species and the closely related Boea hygrometrica. Reverse transcription quantittative PCR analysis confirmed the expression profile of hrh-miR156a-5p and hrh-miR157-5p established by sRNA sequencing and revealed an inverse expression pattern between these miRNAs and their targets in the desiccated tissue. Our study suggests that the miR156/157 and miR399 families are essential for plant survival under severe drought due to their ability to control plant development and growth by modulating transcription factor expression.

Real time quantitative PCR (qPCR) is a powerful tool for studying the expression of specific genes. The accuracy and reliability of qPCR analysis data require the selection of reference genes with stable expression. However, the reference genes that can be used for qPCR of Rehmannia chingii and R. henryi have not yet been identified. In this study, based on the transcriptome data of R. chingii and R. henryi, we initially selected genes with relatively stable expression in different samples. We screened six candidate reference genes in R. chingii and R. henryi and calculated their expression abundance by real time qPCR. Their expression stability was evaluated by three algorithms geNorm, NormFinder, and BestKeeper. Although the results obtained by different algorithms were not completely consistent, R. chingii type 2A phosphatase activator TIP41 and R. chingii 18S ribosomal RNA had the highest expression stability in six different samples of R. chingii, and R. henryi 18S ribosomal RNA and R. henryi actin showed the most stable expression in different samples of R. henryi. In addition, based on transcriptome data, four genes were screened in R. chingii and R. henryi, and the expression stability of the selected reference genes was further verified. This study laid the foundation for further analysis and verification of the functions of important genes in R. chingii and R. henryi.

Bupleurum chinense DC., a source of the traditional Chinese medicine Bupleuri Radix, is rich in triterpenoid saikosaponins with high pharmacological activities. The enzyme β-amyrin synthase (BAS), which converts the precursor 2,3-oxidosqualene to produce the triterpene skeleton, is crucial for the biosynthesis of triterpenoid saponins. In this study, we cloned the full-length sequence of the BAS gene from B. chinense, conducted a bioinformatics analysis, and expressed it in Saccharomyces cerevisiae to investigate its function. The cDNA of β-amyrin synthase (BcBAS, GenBank accession number: MN186093) cloned from aseptic seedlings of B. chinense was 2 307 bp with a 2 286 bp open reading frame coding for 761 amino acids. Phylogenetic analysis suggests that the BcBAS protein was closely related to the BAS proteins from Panax ginseng and Betula platyphylla. as chromatography mass spectrometry analysis showed that the enzymatic product was indeed β-amyrin, the precursor of oleanane type triterpenes. Overall, our findings lay the foundation for in-depth analysis of the biosynthesis pathway of saikosaponins.

Soybean is one of the most important economic crops in the world. However, the salinization of soil results in the decrease of soybean yield as it is only a moderately salt-tolerant crop. We treated three soybean cultivars with low and high concentrations of NaCl. The differentially expressed genes between the control group and the salt treatment group were identified by mRNA sequencing and analyzed by gene ontology and Kyoto encyclopedia of genes and genomes annotations. We performed weighted gene co-expression network analysis on all samples and found genes most related to the phenotype. After verifying the results of differentially expressed genes by quantitative PCR, we finally identified Glyma06G01990, Glyma08G22730, Glyma019G05140, and Glyma06G20160 as key genes affecting the soybean growth under salt stress.

Columnar apple is a valuable resource for genetic improvement of cultivated apples due to its special tree architecture. Strigolactones (SLs) are a novel class of plant hormones controlling shoot branching. The content of SLs is higher in columnar apple than in standard apples. In this study, the members of major gene families involved in SLs biosynthesis and signaling were identified from apple genomic sequences and their expression profiles were characterized in columnar and standard apples using reverse transcription quantitative polymerase chain reactions. In comparison with standard apple, the higher expressions of MORE AXILLARY GROWTH genes MdMAX3-1 and MdMAX4-4 were detected in both buds and shoots of columnar apple but the expression of DWARF gene MdD53-4 showed a lower expression in columnar apple. Overexpression of Columnar gene MdCo31 in tobacco increased SLs content and weakened the inhibition of SLs signal transduction by increasing expression of MAX3 and down-regulating the transcription of D53. Thus MdCo31 could be a strong candidate gene for the control of columnar habit.

Leaf venation and coupled physiological function of wild plants co-evolve during the natural selection. How artificial selection affects leaf vein traits and coordinated physiological functions of main crops are largely unknown. This study examined the changes of leaf vein traits and their correlation with gas exchange of flag leaves and yield in eight wheat genotypes of differing ploidy under the same growing conditions. The results indicate that flag leaf vein density (VLA), major-vein density (VLA_major), and minor-vein density (VLA_minor) decreased whereas the proportion of minor-vein length and interveinal distance between small longitudinal veins (IVD) increased during the polyploidization process, and the major advance occurred from the period from diploids to tetraploids. The VLA, VLA_major, and VLA_minor were closely coordinated with maximum net photosynthetic rate (P_N) and photosynthetic N use efficiency (PNUE), but not with stomatal conductance. The proportion of minor-vein length and IVD were negatively related with P_N and PNUE but positively related with N content per area (N_area) during wheat evolution. A higher proportion of minor-vein length and IVD, and a lower VLA_major in flag leaves along with a larger N_area were largely responsible for the increased yield in modern cultivars. The decreased redundancy of leaf vein density and increased minor-vein proportion in modern cultivars can confer a yield advantage during wheat evolution.