Salt-stress induced changes in the leaf proteome of diploid and tetraploid mandarins with contrasting Na and Cl accumulation behaviour

To understand the genotypic variation of citrus to mild salt stress, a proteomic approach has been carried out in parallel on two citrus genotypes (‘Cleopatra’ and ‘Willow leaf’ mandarins), which differ for Na⁺ and Cl⁻ accumulation, and their cognate autotetraploids (4×). Using two-dimensional electrophoresis approximately 910 protein spots were reproducibly detected in control and salt-stressed leaves of all genotypes. Among them, 44 protein spots showing significant variations at least in one genotype were subjected to mass spectrometry analysis for identification. Salt-responsive proteins were involved in several functions, including photosynthetic processes, ROS scavenging, stress defence, and signalling. Genotype factors affect the salt-responsive pattern, especially that of carbon metabolism. The no ion accumulator ‘Cleopatra’ mandarin genotype showed the highest number of salt-responsive proteins, and up-regulation of Calvin cycle-related proteins. Conversely the ion accumulator ‘Willow leaf’ mandarin showed high levels of several photorespiration-related enzymes. A common set of proteins (twelve spots) displayed higher levels in salt-stressed leaves of 2× and 4× ‘Cleopatra’ and 4× ‘Willow leaf’ mandarin. Interestingly, antioxidant enzymes and heat shock proteins showed higher constitutive levels in 4× ‘Cleopatra’ mandarin and 4× ‘Willow leaf’ mandarin compared with the cognate 2× genotype. This work provides for the first time information on the effect of 8 weeks of salt stress on citrus genotypes contrasting for ion accumulation and their cognate autotetraploids. Results underline that genetic factors have a predominant effect on the salt response, although a common stress response independent from genotype was also found.     

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from ‘Wuzishatangju’ (self-incompatible, SI) and ‘Shatangju’ (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between ‘Wuzishatangju’ and ‘Shatangju’, respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of ‘Wuzishatangju’ and ‘Shatangju’. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of ‘Shatangju’ was approximately 10-fold higher than in anthers of ‘Wuzishatangju’. The highest expression level of CrUBE1 was detected in pistils at 7 days after self-pollination of ‘Wuzishatangju’, which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from ‘Wuzishatangju’ and ‘Shatangju’ were successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was significantly inhibited with increasing of CrUBE1 protein concentrations from ‘Wuzishatangju’.     

Assimilate translocation and expression of sucrose transporter,OsSUT1, contribute to high-performance ripening under heat stress in the heat-tolerant rice cultivar Genkitsukushi

High temperature reduces the grain quality of rice, a situation likely to become more frequent because of global warming. We studied the effects of high-temperature stress on grain quality of heat-tolerant cultivar ‘Genkitsukushi’ and heat-sensitive cultivar ‘Tsukushiroman’. When day/night temperatures were 31/26 °C from heading until maturity, the grain quality of ‘Genkitsukushi’ was rated at the first inspection grade (high quality), whereas ‘Tsukushiroman’ showed a remarkable increase in the percentage of white immature kernels (low quality). Nonstructural carbohydrate content in the stem of ‘Genkitsukushi’ the early maturation was significantly higher than in ‘Tsukushiroman’ and greatly decreased under high temperature. From 14 to 21 days after heading, the expression of the sucrose transporter gene, OsSUT1, was higher in the stem of ‘Genkitsukushi’ grown under high temperature than in ‘Tsukushiroman’. In addition, the expression of OsSUT1 in the grains of ‘Genkitsukushi’ was significantly higher than in ‘Tsukushiroman’ during the ripening period. These results indicate that sugar transport functions more effectively in ‘Genkitsukushi’ than in ‘Tsukushiroman’, and that the effectiveness of sugar transport contributes to maintaining high grain quality in ‘Genkitsukushi’ under high-temperature conditions.     

The complete mitochondrial genome of the leafminer Liriomyza sativae (Diptera: Agromyzidae): great difference in the A+T-rich region compared to Liriomyza trifolii

The complete mitochondrial genome sequence of Liriomyza sativae Blanchard (15,551 bp) was determined and analyzed in this study. The circular genome contained 37 genes including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and an A + T-rich region. The initiation codons of COI and ND1 were ‘ATCA’ and ‘GTG’, respectively. ND2 gene used the truncated termination codon ‘T’. All the tRNA genes had the typical cloverleaf secondary structures except for tRNA^Ser(AGN) gene, which was found with the absence of a DHU arm. In addition, a tRNA-like secondary structure (tRNA^Met) was found in the A + T-rich region. The great difference was that the length of L. sativae A + T-rich region was 597 bp shorter than that of Liriomyza trifolii (Burgess). Meanwhile, some minor differences such as ‘TATA’ block were also observed in L. sativae in contrast to ‘TACA’ block in L. trifolii. There were also some essential structure elements such as ‘TATA’ block, ‘G(A)_nT’ block, poly-T stretch and stem-and-loop structure in the A + T-rich region of L. sativae mitochondrial genome.     

New universal mitochondrial PCR markers reveal new information on maternal citrus phylogeny

The aim of this work was to provide a set of mitochondrial markers to reveal polymorphism and to study the maternal phylogeny in citrus. We first used 44 universal markers previously described in the literature: nine of these markers produced amplification products but only one revealed polymorphism in citrus. We then designed six conserved pairs of primers using the complete mitochondrial DNA sequences of Arabidopsis thaliana and Beta vulgaris to amplify polymorphic intergenic and intronic regions. From these six pairs of primers, three from introns of genes coding for NADH dehydrogenase subunits 2, 5, and 7, revealed polymorphism in citrus. First, we confirmed that citrus have a maternal mitochondrial inheritance in two populations of 250 and 120 individuals. We then conducted a phylogenic study using four polymorphic primers on 77 genotypes representing the diversity of Citrus and two related genera. Seven mitotypes were identified. Six mitotypes (Poncirus, Fortunella, Citrus medica, Citrus micrantha, Citrus reticulata, and Citrus maxima) were congruent with previous taxonomic investigations. The seventh mitotype enabled us to distinguish an acidic mandarin group (‘Cleopatra’, ‘Sunki’ and ‘Shekwasha’) from other mandarins and revealed a maternal relationship with Citrus limonia (‘Rangpur’ lime, ‘Volkamer’ lemon) and Citrus jambhiri (‘Rough’ lemon). This mitotype contained only cultivated species used as rootstocks due to their good tolerances to abiotic stress. Our results also suggest that two species classified by Swingle and Reece, Citrus limon, and Citrus aurantifolia, have multiple maternal cytoplasmic origins.     

Differential responses of two Stellaria longipes ecotypes to ultraviolet-B radiation and drought stress

Enhanced ultraviolet-B (UVB) radiation and water deficit affect plant growth and development. We determined the effects of UVB and drought stress on growth parameters and chemical attributes of two ecotypes (alpine and prairie) of Stellaria longipes under controlled-environment conditions. Clonal ramets of these ecotypes were grown under three UVB levels (0, 5, and 10 kJ m⁻² d⁻¹) and exposed to two watering regimes (well watered and drought stressed) for 21 days. Compared to the alpine, the prairie ecotype was taller, had higher number of nodes, and greater leaf area and specific leaf weight (leaf dry weight: leaf area), which resulted in increased dry matter in this ecotype. Overall, ‘prairie’ was higher in total chlorophyll (Chl), but lower in Chla:b ratio, flavonoids, and ethylene, than ‘alpine’. In both ecotypes, UVB and drought stress reduced growth and dry matter, whereas UVB increased carotenoids and flavonoids. Drought stress decreased ethylene evolution. These characteristics were also determined in plants growing in the field. In the field-growing plants, ‘prairie’ had higher growth and dry matter, but lower Chla:b ratio and flavonoids, than ‘alpine’. The two ecotypes responded differentially to UVB and watering regime, as ‘prairie’ appeared to be more sensitive to UVB and drought stress than ‘alpine’.     

Development of mesenchymal stem cells partially originate from the neural crest

Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells isolated from many adult tissues. Previous studies reported that MSCs can differentiate to both mesodermal and neural lineages by a phenomenon referred to as ‘‘dedifferentiation’’ or ‘‘transdifferentiation’’. However, since MSCs have only been defined in vitro, much of their development in vivo is still unknown. Here, we prospectively identified MSCs in the bone marrow from adult transgenic mice encoding neural crest-specific P0-Cre/Floxed-EGFP and Wnt1-Cre/Floxed-EGFP. EGFP-positive MSCs formed spheres that expressed neural crest stem cell genes and differentiated into neurons, glial cells, and myofibroblasts. Interestingly, we observed MSCs both in the GFP⁺ and GFP⁻ fraction and found that there were no significant differences in the in vitro characteristics between these two populations. Our results suggest that MSCs in adult bone marrow have at least two developmental origins, one of which is the neural crest.     

Involvement of CitCHX and CitDIC in Developmental-Related and Postharvest-Hot-Air Driven Citrate Degradation in Citrus Fruits

Citrate is the predominant organic acid associated with taste in citrus fruit. Although citrate metabolism has been widely studied in recent years, the potential contributions of transport proteins to citrate content remain unclear. In the present study, high-acid citrus fruit Gaocheng (‘GC’, Citrus sp.) and low-acid citrus fruit Satsuma mandarin (‘SM’, Citrus unshiu Marc.) were selected for study, and the degradation of citrate was deduced to be the main cause of the difference in acidity in fully mature fruits. RNA-seq analysis was carried out on ‘GC’ and ‘SM’ fruit samples over the same time course, and the results indicated that citrate degradation occurred mainly through the glutamine pathway, catalyzed by CitAco3-CitGS2-CitGDU1, and also two transport-related genes, CitCHX and CitDIC, were shown to be associated with citrate degradation. These results were confirmed by real-time PCR. In postharvest ‘GC’ fruit, the expressions of these two transport-related genes were induced by 2-fold under hot air treatment, accompanied by a reduction of 7%-9% in total acid degradation. Transient expression of CitCHX and CitDIC in tobacco leaves was performed, and the citrate content was reduced by 62%, 75% and 78% following CitCHX, CitDIC and CitCHX plus CitDIC treatments, respectively, as compared with expression of an empty vector. Overall, these data indicated that two transport proteins, CitCHX and CitDIC, are not only involved in citrate degradation during fruit development, but also involved in postharvest hot air triggered citrate reduction.     

Reference gene selection for real-time quantitative polymerase chain reaction normalization in “Swingle” citrumelo under drought stress

We describe the first systematic evaluation of reference genes for use in real-time quantitative polymerase chain reaction (qPCR) for water deficit stress studies in the citrus rootstock “Swingle” citrumelo. The expression levels of seven reference genes—cyclophilin (CYP), cathepsin (CtP), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), β-tubulin (TUB), and ADP ribosylation factor (ADP)—during drought stress were tested using geNorm and NormFinder programs. Results from four experimental conditions indicated that EF1α and ADP were the most stable reference genes. Relative expression levels of Δ1-pyrroline-5-carboxylate synthetase (P5CS) was used for reference gene validation.     

Economic threshold for Tetranychus urticae (Acari: Tetranychidae) in clementine mandarins Citrus clementina

Tetranychus urticae is a key pest of citrus in Spain, especially of clementine mandarin trees. The effects of this mite on fruit production were assessed in 24 clementine trees for three consecutive years. Trees were visited weekly and spider mite and phytoseiid mite populations and leaf flush patterns were estimated. At the end of the season, mandarins were harvested, weighed, and mite damage (scarring on the fruit) characterized. Negative relationships between spider mite density and yield (kg/tree) and fruit damage (% scarred fruit rind) were found. The multivariate regressions highlighted the key role of phytoseiid mites and leaf flush patterns, which were negatively related to fruit damage. The shortest sampling period that satisfactorily predicted fruit damage at harvest, extended from August to mid-October. For IPM purposes, an action threshold of 31.1 mites m^?2 of symptomatic leaf was estimated. Taking into account spider mite dynamics, the economic threshold ranged from 10 to 15 mites m^?2 of symptomatic leaf. When this threshold is exceeded growers would have a 1-week window to apply the control technologies against T. urticae of their choice.     

Phylogenetic Relationships of Citrus and Its Relatives Based on matK Gene Sequences

The genus Citrus includes mandarin, orange, lemon, grapefruit and lime, which have high economic and nutritional value. The family Rutaceae can be divided into 7 subfamilies, including Aurantioideae. The genus Citrus belongs to the subfamily Aurantioideae. In this study, we sequenced the chloroplast matK genes of 135 accessions from 22 genera of Aurantioideae and analyzed them phylogenetically. Our study includes many accessions that have not been examined in other studies. The subfamily Aurantioideae has been classified into 2 tribes, Clauseneae and Citreae, and our current molecular analysis clearly discriminate Citreae from Clauseneae by using only 1 chloroplast DNA sequence. Our study confirms previous observations on the molecular phylogeny of Aurantioideae in many aspects. However, we have provided novel information on these genetic relationships. For example, inconsistent with the previous observation, and consistent with our preliminary study using the chloroplast rbcL genes, our analysis showed that Feroniella oblata is not nested in Citrus species and is closely related with Feronia limonia. Furthermore, we have shown that Murraya paniculata is similar to Merrillia caloxylon and is dissimilar to Murraya koenigii. We found that “true citrus fruit trees” could be divided into 2 subclusters. One subcluster included Citrus, Fortunella, and Poncirus, while the other cluster included Microcitrus and Eremocitrus. Compared to previous studies, our current study is the most extensive phylogenetic study of Citrus species since it includes 93 accessions. The results indicate that Citrus species can be classified into 3 clusters: a citron cluster, a pummelo cluster, and a mandarin cluster. Although most mandarin accessions belonged to the mandarin cluster, we found some exceptions. We also obtained the information on the genetic background of various species of acid citrus grown in Japan. Because the genus Citrus contains many important accessions, we have comprehensively discussed the classification of this genus.     

Syntheses and structures of some adducts of silver(I) oxyanion salts with some 2-N,O-donor-substituted pyridine bases

Syntheses and room-temperature single crystal X-ray structural characterizations are recorded for a variety of silver(I) oxyanion (perchlorate, nitrate and trifluoroacetate (‘tfa’) (increasing basicity)) adducts, AgX, with a number of pyridine (‘py’) bases, L, functionalized in the 2-position with N- or O-donor groups, namely 2-amino-, 2-amino-6-methyl-, 2-aminomethyl-, 2-hydroxy-, 2-methoxy- and 2-acetyl- pyridines, ‘2np’, ‘nmp’, ‘amp’, ‘ohp’, ‘mop’, and ‘acp’. A variety of stoichiometries and associated structural types are defined: [Ag(chelate)₂]X, L/X = amp,acp/ClO₄, [XAg(chelate)₂], L/X = acp/tfa, of 1:2 AgX:L stoichiometry; for 1:1 stoichiometry, although a discrete mononuclear complex [(chelate)Ag(O₂NO)] is defined for AgNO₃: acp (1:1), all others are polymers, successive silver atoms being linked by N,N′-bridging ligands singly (L/X = 2np/ClO₄ (?HAgHTAgTHAgH?), amp/ClO₄, NO₃ (?HTAgHTAg?) (‘H’ ≡ head, ‘T’ = tail)) or pairwise, ?L₂AgX₂AgL₂Ag? (L/X = 2np/tfa, nmp/NO₃). More complex polymeric arrays are found with L/X = ohp/NO₃, tfa, where interaction with the metal takes place via the O-donor only, the py functionality being protonated, and in adducts of more complex stoichiometry AgNO₃:mop (2:3) and AgNO₃:2np (3:4).     

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72 h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.     

Characterization of the complete mitochondrial genome of Chilo auricilius and comparison with three other rice stem borers

The mitogenome of Chilo auricilius (Lepidoptera: Pyraloidea: Crambidae) was a circular molecule made up of 15,367 bp. Sesamia inferens, Chilo suppressalis, Tryporyza incertulas, and C. auricilius, are closely related, well known rice stem borers that are widely distributed in the main rice-growing regions of China. The gene order and orientation of all four stem borers were similar to that of other insect mitogenomes. Among the four stem borers, all AT contents were below 83%, while all AT contents of tRNA genes were above 80%. The genomes were compact, with only 121–257 bp of non-coding intergenic spacer. There are 56 or 62-bp overlapping nucleotides in Crambidae moths, but were only 25-bp overlapping nucleotides in the noctuid moth S. inferens. There was a conserved motif ‘ATACTAAA’ between trnS2 (UCN) and nad1 in Crambidae moths, but this same region was ‘ATCATA’ in the noctuid S. inferens. And there was a 6-bp motif ‘ATGATAA’ of overlapping nucleotides, which was conserved in Lepidoptera, and a 14-bp motif ‘TAAGCTATTTAAAT’ conserved in the three Crambidae moths (C. suppressalis, C. auricilius and T. incertulas), but not in the noctuid. Finally, there were no stem-and-loop structures in the two Chilo moths.     

Integrative analyses of transcriptome and carotenoids profiling revealed molecular insight into variations in fruits color of Citrus Reticulata Blanco induced by transplantation

Citrus fruits exhibit vivid color and are favored extensively. However, the biochemical and molecular mechanism of Citrus Reticulata Blanco fruits coloring, especially the effect of transplantation on fruits coloring, is unclear. Herein, RNA-Seq and carotenoids profiling were applied to investigate the effect of transplantation on Orah mandarin fruits coloring. Transplantation induces fruit color shallowing, Ca²⁺ and ACC level declining and IAA level increasing. Transplantation induced variation in fruit skin and pulp carotenoids, mainly β-citraurin as one of the important pigments of citrus peel. 2253 up-regulated genes, 1103 down-regulated genes in skin and 815 up-regulated genes, 534 down-regulated genes in pulp of transplanted tree fruits were identified by RNA-Seq. The DEGs involved hormone signal, carotenoids biosynthesis and TFs such as MYB and bHLH family TFs. The carotenoid cleavage dioxygenase gene (Ciclev10028113m.g) is positively correlated with β-citraurin and regulated directly and/or indirectly by MYB1R1, PIF4, ACC and IAA. Integrative analyses revealed potential molecular insights into Orah mandarin peel color variation during transplantation.