MRCTLH (muskelin/RanBPM/CTLH) is a protein complex found in humans (MRCTLH) that is involved in the regulation of numerous cellular processes, such as gluconeogenesis, cell signaling, development, nuclear extrusion, cell morphology, or stability of different proteins. According to genomic data, all eukaryotes have similar protein complexes. In yeast, a similar protein complex named GID was found to be involved in the regulation of gluconeogenesis. LICC1 is a maize protein whose sequence resembles that of TWA1 in humans and GID8 in yeast, which are central components of the MRCTLH and GID complexes. LICC1 contains three highly conserved protein domains, LisH, CTLH, and CRA, typical of this protein family. Twa1 and gid8 are unique genes in human and yeast genomes. However, three copies of licc1 are present in the maize genome and multiple copies are present in other plant genomes. This result suggests the presence of multiple variants of the MRCTLH/GID complex in plants, which could increase its regulatory capacity. We also demonstrate here that LICC1 has the ability to interact with microtubules, similarly to the human TWA1. This interaction reinforces the idea that the LICC1 protein from maize, and its homologues in plants and, in general, the GID/MRCTLH complex in plants, can perform biological functions similar to those in humans and yeast.

The aim of this study was to evaluate the effects of melatonin on photoinhibition under chilling stress in cucumber seedlings and to inquire into any mechanisms of mitigation. Under chilling stress, the net photosynthetic rate declined dramatically but the decline was significantly mitigated by irrigation with a melatonin solution. Possible mechanisms for this mitigation are that melatonin accelerates xanthophyll de-epoxidation by upregulating the transcription of the violaxanthin de-epoxidase gene (CsVDE) and down-regulating that of the zeaxanthin cyclase gene (CsZE) during chilling. There was also a rise in non-photochemical quenching (NPQ) if seedlings were pretreated with melatonin before chilling. The efficient operation of the xanthophyll cycle helped consume excessive excitation energy in photosystem (PS) Ⅱ and so protected the photosynthetic system. Melatonin also modulated the partitioning of absorbed excitation energy in PS Ⅱ as evidenced by alleviation of the decrease in quantum yield of photochemical energy conversion in PS Ⅱ under chilling stress, by alleviation of the rise in quantum yield of non-regulated, non-photochemical energy loss in PS II and by increasing the regulated non-photochemical energy loss in PS II. This study presents a new understanding of the mechanisms through which melatonin mitigates photoinhibition by modulating the partitioning of absorption energy in PS Ⅱ based on the xanthophyll cycle.

Masson pine (Pinus massoniana Lamb.) is an important tree species of high economic value in southern China, but osmotic stress threatens its growth and development. In this study, physiological measurements and RNA-Seq analysis were used to clarify the physiological and molecular responses of P. massoniana under osmotic stress. Osmotic treatment caused cell membrane damage and reactive oxygen species (ROS) accumulation in the tree seedlings, but it also increased their antioxidant enzyme (superoxide dismutase, peroxidase, and catalase) activities and osmotic substances (soluble sugars, proline, and trehalose) content so as to adjust to osmotic stress conditions. A total of 1 789 differentially expressed genes (DEGs) were identified by transcriptome sequencing, of which 962 were up-regulated and 827 genes down-regulated. A series of stress-induced genes associated with signal transduction, ROS-scavenging, osmotic regulation, late embryogenesis abundant (LEA) protein, pentatricopeptide repeat-containing protein, and transcription factors' regulation were distinguishable. This detailed investigation of the stress-responsive genes and pathways provides new insight into molecular mechanism of abiotic stress response in P. massoniana. Further, this study's data can contribute to genetic engineering or molecular breeding efforts to enhance osmotic resistance in P. massoniana stands.

The myeloblastosis (MYB) transcription factors are essential for plant stress responses. They can enhance plant tolerance to abiotic stresses (e.g., drought, salinity, and cold) via improved physiological and biochemical responses including the accumulation of metabolites. In this study, we constructed a Panax ginseng MYB4 (PgMYB4) gene expression vector and established the stable transgenic Arabidopsis thaliana lines to study the effects of this gene on plant stress tolerance. The germination rate and seedling taproot length were greater for the PgMYB4-overexpressing plants than for the wild-type plants. Accordingly, the overexpression of PgMYB4 in Arabidopsis enhanced seedling tolerance to drought, salt, and cold conditions. Under drought stress, the relative chlorophyll content decreased less, the proline content increased more, and the water loss rate decreased more in the transgenic plants than in the wild type. The expressions of stress-related genes responsive to dehydration 19A, responsive to dehydration 22, responsive to desiccation 29A, cold-regulated 15A, cold-regulated 47, and pyrroline-5-carboxylate synthase 1 were significantly upregulated in the transgenic Arabidopsis plants. Under high salt stress, the kinesin 1 (KIN1) expression was significantly upregulated in the transgenic plants. In response to the low temperature stress, the dehydration-responsive element binding protein 2A and KIN1 expressions increased dramatically in the transgenic Arabidopsis plants. Thus, PgMYB4 positively regulated the stress tolerance gene networks, which promoted the expression of anti-stress effector genes. This gene may be useful for ginseng breeding programs aiming to develop new cultivars with enhanced stress tolerance.

Atractylodes lancea (Thunb.) DC has been used widely as a medicinal herb for centuries and is now being used to treat COVID-19 pneumonia. Terpenoids are thought to be its main pharmacologically active constituents. However, their biosynthesis remains uncharacterized in this species. In this study, the terpene synthase gene AlTPS1 was cloned and functionally characterized. We found that AlTPS1 was a bifunctional enzyme that catalyzed the conversion of farnesyl diphosphate to nerolidol and geranyl diphosphate to linalool in vitro. However, it functioned only in the nerolidol production in vivo by transient expression of the AlTPS1 gene in Nicotiana benthamiana leaves maybe due to subcellular compartmentalization of the AlTPS1 in the cytosol. Furthermore, AlTPS1 was highly expressed in leaves, considered to be the sites of nerolidol synthesis. This study is the first in which the cloning and expression of the AlTPS1 gene from A. lancea were analyzed, and it has provided new insights into terpene biosynthesis in A. lancea.

Cannabis sativa, an annual herbaceous plant, produce wide variety of secondary metabolites among which delta-9-tetrahydrocannabinol (THC) is the most important one. The dissection of biosynthetic pathway(s) of this compound and its regulation by transcription factors (TFs) is an important prerequisite for efficient biotechnological manipulation of its secondary metabolome. A polyketide synthase (PKS) of C. sativa catalyzes the first step of cannabinoid biosynthesis, leading to the biosynthesis of olivetolic acid. Cloning and analysis of PKS promoter based on online PLACE, PlantCARE, and Genomatix Matinspector professional databases, indicated that PKS promoter consisted of cis-elements such as TATA-box, CAAT-box, W-box, Myb-box, E-box, and P-box. Plant expression vector PKS::GUS was constructed in such a way that the ATG of the PKS gene was in the frame with the β-glucuronidase (GUS) coding region. Using a combinatorial transient GUS expression system in Nicotiana benthamania leaves, it was shown that heterologous TFs such as HlWRKY1, HlMYB3, HlWDR1 and HlbZIP1 from Humulus lupulus significantly activated PKS promoter. Moreover, Tombusvirus p19 core protein, which is known for silencing suppressor functions, acted in our combinatorial transient expression system as an enhancer of PKS promoter activity along with hop TFs. Our analyses suggested the involvement of the hop derived TFs (HlWRKY1, HlMYB3, HlWDR1 and HlbZIP1A) and p19 in the activation of PKS gene promoter, which could be used for the genetic manipulation of C. sativa to enhance the cannabinoid production.

The GRAS transcription factors encoding proteins ranging from 400 to 700 residues are recognized by their conserved C terminus. Here, a set of 23 CmGRAS genes was identified from a scan of the chrysanthemum (Chrysanthemum morifolium) transcriptome. A phylogenetic analysis implied that nine of these genes could be assigned orthologs to the GRAS gene family, and that four of them formed two pairs of paralogs. A phylogenetic analysis of the GRAS protein family based on the chrysanthemum and recent study of eight representative species of angiosperms showed that most of the CmGRAS genes belonged to a recognized sub-group. CmGRAS4 and CmGRAS10 were strongly transcribed in flowers and roots, respectively. The DELLA subfamily transcript abundance of the CmGRAS19 and CmGRAS20 was high in the reproductive tissues and they were responsive to phytohormones and stresses. Establishment of the orthology relationships between the known representative GRAS genes and CmGRAS, and transcriptional profiles of CmGRASs after phytohormone treatments or stresses will facilitate subsequent functional analyses in the GRAS gene family.

The gene NCED1 encodes 9-cis-epoxycarotenoid dioxygenase, which catalyzes oxidative cleavage of 9-cis-epoxycarotenoids neoxanthin and violaxanthin to xanthoxin, a key step in the biosynthesis of abscisic acid in higher plants. In the present study, the complete NCED1 of 1 917 bp was cloned and characterized from rice (Oryza sativa L. cv. N22) as no earlier reports were available for its characterization from indica cultivar. The NCED1 had no intron and encoded a protein of 639 amino acids with a predicted molecular mass of 68.62 kD and pI of 6.07. The aliphatic index and grand average of hydropathicity were found to be 77.04 and -0.148, respectively. Multiple alignment analysis revealed that the sequence shared a high identity with the Oryza sativa japonica group (100 %) followed by Triticum aestivum (90 %), Hordeum vulgare (90 %), and Zea mays (89 %). The enzyme had a RPE65 domain of 476 amino acid residues. The RPE65 domain requires Fe(II) as a cofactor coordinated with 4 histidine residues and 3 glutamic acid residues. The phylogenic tree shows that NCED1 of japonica rice and NCED1 of indica rice were in the same group. They might have been evolved from a common ancestor. Analysis with a PSORT III tool shows that NCED is a chloroplastic protein. The real-time quantitative PCR and RNA-sequencing studies show that the expression of NCED1 was progressively reduced with increasing water stress, and a negative correlation between expression of OsNCED1 and severity of stress was established. Further, NCED1 expression negatively correlated with abscisic acid (ABA) accumulation under water stress whereas in some other species its expression increased along with ABA accumulation. This might be due to feedback inhibition of the ABA biosynthesis in rice.

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.

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.

Three genes encoding novel D-hordeins, Ns 1.3, Ns 2.6, and Ns 2.9 were isolated from Psathyrostachys juncea. The Ns 1.3 differed from Ns 2.6 and Ns 2.9 by having a shorter open reading frame (< 1.5 kb versus > 2.5 kb), and was probably not expressed as a normal protein, while the activities for Ns 2.6 and Ns 2.9 were verified by bacterial expression. Though highly similar primary structure to wheat high molecular mass glutenin subunits (HMM-GSs) and barley D-hordeins, Ns 2.6 and Ns 2.9 had more cysteine residues (nine in total) and a larger molecular mass than HMMGSs, and a longer N-terminal length than D-hordeins. Phylogenetic analysis revealed that the Ps. juncea D-hordeins were divided into Ns 1.3 type and Ns 2.6/Ns 2.9 type. Divergence times indicated that Ns 1.3 diverged the earliest from the orthologous Triticeae locus, while Ns 2.6 and Ns 2.9 and the D-hordeins from two Hordeum species diverged nearly at the same time from those loci, and the divergence between the D-hordeins of H. chilense and Ns 2.6/Ns 2.9 was more recent than between the two Hordeum species. The novel Ps. juncea D-hordeins have the potential to be very important for improving the end-use quality of wheat flours because of the presence of extra cysteine residues and longer repetitive domain, in addition they can contribute to the understanding of the evolution of Triticeae prolamins.

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.

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.

Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing method used for unraveling gene functions. As an attractive alternative to mutant collections or stable transgenic plants, it has been widely used in reverse-genetics studies owing to its ease use and quick turnaround time. Turnip yellow mosaic virus (TYMV) has the ability to induce VIGS in Arabidopsis thaliana. However, the conventional vector construction is difficult and the efficiencies of the infection methods are low. Here, we improved the vector construction and viral infection methods, inserted an inverted-repeat fragment of the phytoene desaturase gene into a TYMV-derived vector by homologous recombination and transformed Brassica rapa with plasmid DNA harboring a cDNA copy of the TYMV genome through particle bombardment. An apparent photobleaching phenotype was detected and efficient VIGS was induced. An 80-bp fragment was sufficient to produce VIGS in leaves, stems, roots, flowers, siliques, and stalks of B. rapa. Because TYMV has a wide host range in Brassica, the VIGS system described here will contribute to the improvement of high-throughput technology and efficient functional research in B. rapa and other Brassicaceae crops.

Salicylic acid (SA) is an important plant hormone involved in the activation of defense responses against environmental stresses. However, there are still large of unsolved mysteries about how SA pretreatment affects the establishment of plant stress tolerance. In this study, application of SA at different concentrations and different times were conducted to investigate their effects on the response of Arabidopsis seedlings to salt stress. The pretreatment with 10 or 20 μM SA for more than 6 h promoted Arabidopsis seedlings resistance to salt stress. On the other hand, pretreatment with 200 μM SA reduced Arabidopsis resistance to salt stress and aggravated oxidative damage to the seedlings. At all concentrations used, SA pretreatment inhibited the total respiration and promoted reactive oxygen species (ROS) generation. However, the ROS content in 10 or 20 μM SA pretreated seedlings decreased to the basal level within 6 h and high activities of antioxidant enzymes and alternative oxidase were maintained. Notably, the SA-enhanced salt stress resistance was significantly impaired by blocking alternative oxidase (AOX) pathway. Our findings indicate that SA-mediated salt stress response is in a dose- and time-dependent manner and that the effects were related to the induction of AOX capacity and antioxidant system.

To study the biological function of activity of bcl complex (ABC1) proteins in Lepidium apetalum Willd., genes encoding ABC1 family proteins were identified from the seed transcriptome. The sequence most closely related to germination at a low temperature was selected and gene expressions in response to low temperature stress further studied. The results show that 21 ABC1 genes were expressed in seeds germinating at the low temperature: 4 genes were upregulated, 6 were downregulated, and 11 were not significantly different from controls. The results of fluorescence quantification of the low-temperature stress on the seedlings of 7-d-old L. apetalum showed that seven genes were up-regulated, six genes were down-regulated, and eight genes had no significant difference. Real-time quantitative PCR results show that under the low temperature stress, the expression of the LaAbc1-3 gene increased, but its expression decreased after some time. The expression of this gene increased again after removing the low temperature stress. The expression of LaAbc1-21 gene in L. apetalum seedlings showed a trend of decreasing first and then increasing. The LaAbc1-3 gene was insensitive to salt stress. Expression of the LaAbc1-21 gene was significantly up-regulated during the salt stress. Under osmotic stress, the expression of the LaAbc1-3 gene was down-regulated, and the expression was negatively correlated with polyethylene glycol (PEG-6000) concentration. Under the PEG-6000 treatment, the expression of the LaAbc1-21 gene was significantly up-regulated, and the expression was positively correlated with concentration. These results provide a basis for further analysis of the role of the ABC1 genes in the stress resistance of L. apetalum.

Festulolium are grasses formed through interspecific hybridisation of ryegrass (Lolium) and fescue (Festuca) species. The Lolium-Festuca genome complex represents a vast array of heterogeneous and largely outbreeding grass species that have evolved, diverged, and adapted, allowing their world-wide colonisation of temperate grasslands. While strategies for grass improvement have focused primarily on intraspecific breeding and, in particular, on the agronomically desirable species Lolium perenne and Lolium multiflorum, a growing interest has emerged in interspecific hybrids as alternatives. The principal driver has been the increased appreciation of the capability of wide hybridisation to extend phenotypic variation beyond the ranges available within a single species. Lolium and Festuca species share complementary and desirable traits, and the prime aim in Festulolium (Festuca × Lolium) cultivar development has been to combine the agronomic performance of Lolium and the stress resistance of Festuca species. Advances in Festulolium development are timely, and support strategies aimed at delivering a more sustainable future for livestock agriculture, with grass cultivars that are persistent and productive. Festulolium hybrids occur naturally, including examples that demonstrate extreme heterosis with adaptations sufficient to sustain growth in harsh conditions. However, they are largely sterile and their perpetuity depends mainly on vegetative propagation. Synthetic Festulolium hybrids suitable for plant breeding require genome stability and fertility, sufficient for a cost-effective seed production. To this end, suitable amphiploid and introgression-breeding approaches have been developed. Herein, we provide detailed selected highlights in the research and breeding of Festulolium. In addition, recognising the multifunctional properties of grasslands and the development of enabling technologies that permit their study, we review additional benefits likely to accrue from Festulolium that may mitigate climate change effects and provide valuable ecosystem services.

Cytosine methylation plays an important role in plant development by regulating gene expressions. However, few studies have investigated methylation changes during the tissue differentiation and development of perennial plants. Here, the fluorescence-labeled methylation-sensitive amplified polymorphism method was used with eight primer combinations to detect methylation in leaves and xylem obtained at the stages of inflorescence emergence (IE), ovary start growth, and fruit maturity (FM) in two pecan (Carya illinoinensis) cvs. Pawnee and Stuart. The results show that the total methylation in the xylem was generally higher than in the leaves at each stage. Substantial methylation variations were observed at the amplified sites in pecan tissues at the various stages. The methylation patterns changed between the leaf and xylem, with frequencies from 44.97 to 67.01 % over the three stages in the two cultivars, among which the variation frequency between the tissues at the FM stage was the highest for each cultivar. The frequencies of methylation variation between the leaf samples at any two stages ranged from 31.86 to 45.88 %, with higher variation frequencies between the xylem samples (40.90 - 59.44 %) for each cultivar, which is consistent with the comparative results of polymorphism rates between the leaf and xylem over the three stages. Cluster analysis and principal coordinate analysis suggest that the xylem at the IE and FM stages had relatively distant epigenetic relationships with other tissue samples as a whole. This study reveals the patterns of methylation variation and methylation relationships among pecan tissues undergoing different developmental processes, implying the important roles of methylation in tissue differentiation and development of trees. These results lay a theoretical foundation for elucidating the regulatory mechanisms of methylation involved in tree development.

Zinc deficiency is a worldwide problem for crops including potato (Solanum tuberosum L.), the fourth most important crop worldwide. The zinc/iron-regulated transporter-like protein (ZIP) transporter family is thought to play key roles in Zn uptake and transport. However, little is known about the potato ZIP family. In this study, 12 genes encoding members of the ZIP family were identified in the potato genome. The 12 StZIP genes were predicted to encode proteins of 220 - 407 amino acids harboring 5 - 9 putative transmembrane domains (TMDs), and 11 of these proteins had a variable region rich in histidine residues between TMDIII and TMDIV. A phylogenetic analysis divided the StZIPs into four groups on the basis of gene structure and conserved motifs. Furthermore, the StZIP expression profiles were determined under Zn-deficiency in both high and low Zn-content genotypes. Four differentially expressed genes, StZIP6, -9, -11, and -12, were identified in tubers of the two genotypes under Zn-deficiency, and StZIP11 and StZIP12 may have a more prominent function in Zn uptake and accumulation in potato tubers owing to their higher expressions. Thus, the results provide useful information for further studying the functions of StZIP genes.

To study the biological function of late embryogenesis abundant (LEA) proteins in Lepidium apetalum Willd., genes encoding LEA family proteins were identified from the seed transcriptome. The sequence most closely related to germination at a low temperature was selected and gene expressions in response to low temperature stress further studied. The results show that 27 LEA genes were expressed in seeds germinating at the low temperature: 3 genes were upregulated, 20 were downregulated, and 4 were not significantly different from controls. The most prominent of the upregulated genes, LaLEA1, contained an open reading frame of 624 bp and encoded 208 amino acids. The protein was rich in hydrophilic amino acids including threonine, alanine, glutamine, and lysine. It is predicted that the secondary structure contains α-helices and irregular curls. Real-time quantitative PCR results show that under low temperature stress, the expression of LaLEA1 was first downregulated and then upregulated rapidly, reaching its highest expression at 12 h, then the expression of LaLEA1 was reduced slightly but maintained higher than that of the non-stress group. As the expression of LaLEA1 was significantly altered in response to low temperature stress, we investigated the expression of LaLEA1 also in response to other abiotic stresses, i.e., salinity and drought. L. apetalum seedlings wilted in the early stage following NaCl or osmotic (polyethylene glycol) stresses, but recovered quickly, showing a strong tolerance. Real-time quantitative PCR results show that LaLEA1 was rapidly upregulated following salt and osmotic stresses, and its expression profile was closely related to NaCl or PEG concentrations. Expression was up to 7.9-fold higher than that of the control after 6 h of salt stress. These results suggest that L. apetalum seedlings responded quickly to salt stress. The response to osmotic stress was slightly slower; expression of LaLEA1 was 6.0-fold higher than that of the control after 12 h. Thus, LaLEA1 played an important role in abiotic stress tolerance. These results provide a basis for further analysis of the role of the LEA genes in the stress resistance of L. apetalum.

γ-Aminobutyric acid (GABA) regulates plant tolerance to abiotic stresses; however, a transcriptomic change and key stress-related genes induced by GABA have not been investigated in plants during a prolonged period of salt stress. Roots of creeping bentgrass (Agrostis stolonifera) cv. Penncross were pretreated with or without 0.5 mM GABA solution for 2 days and then subjected to salt stress for 20 days (150 mM NaCl solution for 3 d, 200 mM NaCl for another 3 d, and 250 mM NaCl for 14 d) in controlled growth chambers. The application of GABA significantly increased GABA content in roots and alleviated a salt-stress induced decrease in GABA content in leaves. This was associated with a significant increase in salt tolerance as demonstrated by a significantly higher leaf relative water content, photochemical efficiency, performance index on absorption basis, and lower electrolyte leakage in GABA-pretreated plants as compared to untreated plants under salt stress. Transcriptomic analysis found that GABA-induced salt tolerance was closely associated with saccharide, amino acid, and lipid metabolism. The GABA upregulated key differentially expressed genes including cytochrome P450 (CYP450), zinc transporter 29 (ZTP29), alpha-amylase 3 (AMY3), 3-ketoacyl-CoA synthase 6 (KCS6), aldehyde oxidase (AO), acetyl-CoA carboxylase 1 (ACC1), and magnesium-chelatase (Mg-CHT) involved in zinc homeostasis, starch degradation, and the biosynthesis of wax, fatty acid, chlorophyll, and abscisic acid, which could contribute to GABA-regulated salt tolerance. Current findings prove that GABA application is an efficient approach to enhance salt tolerance of creeping bentgrass during a prolonged period of salt stress and also provide valuable information to better understand key candidate genes and regulatory pathways of GABA-induced salt tolerance in plants.

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.

Plants are severely affected by many biotic stresses, which cause a reduction in crop quality and quantity. One of the strategies to manage biotic stresses is the generation of transgenic plant lines that can be used as biosensors. These biosensor plants can trigger an early warning upon any pathogen infection. Two promoters with β-glucuronidase reporter gene fusions were constructed. The first contained the flagellin sensing 2 gene promoter, whereas the second contained synthetic promoter containing four repeats of cis-acting elements from the pathogen-related protein 1 gene and two transcription enhancers from the 35S promoter. Transformed leaves were treated with a phytohormone salicylic acid to mimic the occurrence of biotic stress. Validation of reporter gene expression induced from both constructs in transformed potato leaves displayed an increase upon salicylic acid treatment. The results reflect that both constructs could serve in the production of potato biotic stress biosensors.