ERF108 from Poncirus trifoliata (L.) Raf. functions in cold tolerance by modulating raffinose synthesis through transcriptional regulation of PtrRafS.

Ethylene-responsive factors (ERFs) are plant-specific transcription factors involved in cold stress response, and raffinose is known to accumulate in plants exposed to cold. However, it remains elusive whether ERFs function in cold tolerance by modulating raffinose synthesis. Here, we identified a cold-responsive PtrERF108 from trifoliate orange (Poncirus trifoliata (L.) Raf.), a cold-tolerant plant closely related to citrus. PtrERF108 is localized in the nucleus and has transcriptional activation activity. Overexpression of PtrERF108 conferred enhanced cold tolerance of transgenic lemon, whereas virus-induced gene silencing (VIGS)-mediated knockdown of PtrERF108 in trifoliate orange greatly elevated cold sensitivity. Transcriptome profiling showed that PtrERF108 overexpression caused extensive reprogramming of genes associated with signaling transduction, physiological processes and metabolic pathways. Among them, a raffinose synthase (RafS)-encoding gene, PtrRafS, was confirmed as a direct target of PtrERF108. RafS activity and raffinose content were significantly increased in PtrERF108-overexpressing transgenic plants, but prominently decreased in the VIGS plants under cold conditions. Meanwhile, exogenous replenishment of raffinose could recover the cold tolerance of PtrERF108-silenced plants, whereas VIGS-mediated knockdown of PtrRafS resulted in cold-sensitive phenotype. Taken together, the current results demonstrate that PtrERF108 plays a positive role in cold tolerance by modulation of raffinose synthesis via regulating PtrRafS. Our findings reveal a new transcriptional module composed of ERF108-RafS underlying cold-induced raffinose accumulation in plants.

The vascular targeted citrus FLOWERING LOCUS T3 gene promotes non-inductive early flowering in transgenic Carrizo rootstocks and grafted juvenile scions.

Shortening the juvenile stage in citrus and inducing early flowering has been the focus of several citrus genetic improvement programs. FLOWERING LOCUS T (FT) is a small phloem-translocated protein that regulates precocious flowering. In this study, two populations of transgenic Carrizo citrange rootstocks expressing either Citrus clementina FT1 or FT3 genes under the control of the Arabidopsis thaliana phloem specific SUCROSE SYNTHASE 2 (AtSUC2) promoter were developed. The transgenic plants were morphologically similar to the non-transgenic controls (non-transgenic Carrizo citrange), however, only AtSUC2-CcFT3 was capable of inducing precocious flowers. The transgenic lines produced flowers 16 months after transformation and flower buds appeared 30-40 days on juvenile immature scions grafted onto transgenic rootstock. Gene expression analysis revealed that the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and APETALA1 (AP1) were enhanced in the transgenics. Transcriptome profiling of a selected transgenic line showed the induction of genes in different groups including: genes from the flowering induction pathway, APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family genes, and jasmonic acid (JA) pathway genes. Altogether, our results suggested that ectopic expression of CcFT3 in phloem tissues of Carrizo citrange triggered the expression of several genes to mediate early flowering.

PtrCDPK10 of Poncirus trifoliata functions in dehydration and drought tolerance by reducing ROS accumulation via phosphorylating PtrAPX.

Calcium-dependent protein kinases (CDPKs) are important calcium signaling components that have been shown to play crucial roles in modulating plant abiotic stress responses. However, the physiological and regulatory roles of most CDPKs are still poorly understood. Here, we report the functional characterization of PtrCDPK10 from trifoliate orange (Poncirus trifoliata (L.) Raf.) in dehydration and drought stress tolerance. PtrCDPK10, categorized in the Type III subgroup of the CDPK family, was localized to the nucleus and plasma membrane. Transcript levels of PtrCDPK10 were up-regulated by dehydration, salt and ABA treatments. Transgenic trifoliate orange plants overexpressing PtrCDPK10 showed enhanced dehydration tolerance compared with the wild type (WT), whereas VIGS (virus-induced gene silencing)-mediated knockdown of PtrCDPK10 resulted in elevated susceptibility to dehydration and drought stresses. Yeast two-hybrid screening identified several proteins that interacted with PtrCDPK10, including an ascorbate peroxidase (PtrAPX). PtrCDPK10 was shown to phosphorylate PtrAPX based on an in vitro kinase assay. PtrCDPK10-overexpressing transgenic lines exhibited higher PtrAPX mRNA abundance and APX activity and accumulated dramatically less ROS in comparison with the WT, while PtrCDPK10-silenced VIGS lines showed decreased PtrAPX expression and increased ROS level. Taken together, these results demonstrate that PtrCDPK10 promotes dehydration and drought tolerance by, at least in part, phosphorylating APX to modulate ROS homeostasis.

PpCRN7 and PpCRN20 of Phythophthora parasitica regulate plant cell death leading to enhancement of host susceptibility.

BACKGROUND: Phytophthora species secrete cytoplasmic effectors from a family named Crinkler (CRN), which are characterised by the presence of conserved specific domains in the N- and C-terminal regions. P. parasitica causes disease in a wide range of host plants, however the role of CRN effectors in these interactions remains unclear. Here, we aimed to: (i) identify candidate CRN encoding genes in P. parasitica genomes; (ii) evaluate the transcriptional expression of PpCRN (Phytophthora parasitica Crinkler candidate) during the P. parasitica interaction with Citrus sunki (high susceptible) and Poncirus trifoliata (resistant); and (iii) functionally characterize two PpCRNs in the model plant Nicotiana benthamiana. RESULTS: Our in silico analyses identified 80 putative PpCRN effectors in the genome of P. parasitica isolate 'IAC 01/95.1'. Transcriptional analysis revealed differential gene expression of 20 PpCRN candidates during the interaction with the susceptible Citrus sunki and the resistant Poncirus trifoliata. We have also found that P. parasitica is able to recognize different citrus hosts and accordingly modulates PpCRNs expression. Additionally, two PpCRN effectors, namely PpCRN7 and PpCRN20, were further characterized via transient gene expression in N. benthamiana leaves. The elicitin INF-1-induced Hypersensitivity Response (HR) was increased by an additive effect driven by PpCRN7 expression, whereas PpCRN20 expression suppressed HR response in N. benthamiana leaves. Despite contrasting functions related to HR, both effectors increased the susceptibility of plants to P. parasitica. CONCLUSIONS: PpCRN7 and PpCRN20 have the ability to increase P. parasitica pathogenicity and may play important roles at different stages of infection. These PpCRN-associated mechanisms are now targets of biotechnological studies aiming to break pathogen's virulence and to promote plant resistance.

Full genome characterization of 12 citrus tatter leaf virus isolates for the development of a detection assay.

Citrus tatter leaf virus (CTLV) threatens citrus production worldwide because it induces bud-union crease on the commercially important Citrange (Poncirus trifoliata × Citrus sinensis) rootstocks. However, little is known about its genomic diversity and how such diversity may influence virus detection. In this study, full-length genome sequences of 12 CTLV isolates from different geographical areas, intercepted and maintained for the past 60 years at the Citrus Clonal Protection Program (CCPP), University of California, Riverside, were characterized using next generation sequencing. Genome structure and sequence for all CTLV isolates were similar to Apple stem grooving virus (ASGV), the type species of Capillovirus genus of the Betaflexiviridae family. Phylogenetic analysis highlighted CTLV's point of origin in Asia, the virus spillover to different plant species and the bottleneck event of its introduction in the United States of America (USA). A reverse transcription quantitative polymerase chain reaction assay was designed at the most conserved genome area between the coat protein and the 3'-untranslated region (UTR), as identified by the full genome analysis. The assay was validated with different parameters (e.g. specificity, sensitivity, transferability and robustness) using multiple CTLV isolates from various citrus growing regions and it was compared with other published assays. This study proposes that in the era of powerful affordable sequencing platforms the presented approach of systematic full-genome sequence analysis of multiple virus isolates, and not only a small genome area of a small number of isolates, becomes a guideline for the design and validation of molecular virus detection assays, especially for use in high value germplasm programs.

Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones.

Nutrient deficiency has economic and ecological repercussions for citrus fruit crops worldwide. Citrus crops rely on fertilization to maintain good fruit output and quality, whereas new crop management policy aims to reduce fertilizers input. New rootstocks are needed to meet to this constraint, and the use of new tetraploid rootstocks better adapted to lower nutrient intake could offer a promising way forward. Here we compared physiological, biochemical and anatomic traits of leaves in diploid (2x) and doubled-diploid (4x) Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) and Volkamer lemon (Citrus limonia Osb.) seedlings over 7 months of nutrient deficiency. Photosynthetic parameters (Pnet, Gs and Fv/Fm) decreased, but to a lesser extent in 4x genotypes than 2x. Degradation of the ultrastructural organelles (chloroplasts and mitochondria) and compound cells (thylakoids and starches) was also lower in 4x genotypes, suggesting that tetraploidy may enhance tolerance to nutrient deficiency. However, leaf surface (stomata, stomatal density and epithelial cells) showed no nutrient deficiency-induced change. In 4x Citrumelo 4475, the higher tolerance to nutrient deficiency was associated with a lower MDA and H2O2 accumulation than in the 2x, suggesting a more efficient antioxidant system in the 4x genotype. However, few differences in antioxidant system and oxidative status were observed between 2x and 4x Volkamer lemons.

Host-plant resistance associated with Poncirus trifoliata influence oviposition, development and adult emergence of Diaphorina citri (Hemiptera: Liviidae).

BACKGROUND: The Asian citrus psyllid, Diaphorina citri Kuwayama, is the primary vector of the phloem-inhabiting bacterium Candidatus Liberibacter asiaticus putatively responsible for citrus greening (huanglongbing), a devastating citrus disease. Infestations of D. citri frequently develop on Citrus and other genera within the Rutaceae subfamily Aurantioideae including Murraya and Bergera. The genotype Poncirus trifoliata is also a member of the Aurantioideae and readily hybridizes with Citrus spp., but colonization by D. citri is reduced on this genotype. RESULTS: Working with young potted seedlings grown in a greenhouse, we found that the development of D. citri immatures on four P. trifoliata cultivars, especially 'Kryder 55-5', was slower compared with the development of immatures on the susceptible Citrus macrophylla. In choice assays, adult psyllids exhibited antixenotic behavior towards accessions of P. trifoliata and laid fewer eggs on this genotype compared with C. macrophylla. CONCLUSIONS: Based on reduced oviposition and delays in development, P. trifoliata exhibits a combination of antixenosis and antibiosis host-plant resistance to D. citri. A companion plant assay showed that the presence of C. macrophylla stimulated higher oviposition rates on P. trifoliata, but nymph development remained retarded on P. trifoliata. Here, we show that the antixenosis associated with trifoliate accessions can be overcome to some extent by the presence of a preferred susceptible host plant; but in combination with antibiosis P. trifoliata remains an inferior host plant. © 2018 Society of Chemical Industry.

Enhanced ROS scavenging and sugar accumulation contribute to drought tolerance of naturally occurring autotetraploids in Poncirus trifoliata.

Tetraploids have been reported to exhibit increased stress tolerance, but the underlying molecular and physiological mechanisms remain poorly understood. In this study, autotetraploid plants were identified by screening natural seedlings of trifoliate orange (Poncirus trifoliata). The tetraploids exhibited different morphology and displayed significantly enhanced drought and dehydration tolerance in comparison with the diploid progenitor. Transcriptome analysis indicated that a number of stress-responsive genes and pathways were differentially influenced and enriched in the tetraploids, in particular those coding for enzymes related to antioxidant process and sugar metabolism. Transcript levels and activities of antioxidant enzymes (peroxidase and superoxide dismutase) and sucrose-hydrolysing enzyme (vacuolar invertase) were increased in the tetraploids upon exposure to the drought, concomitant with greater levels of glucose but lower level of reactive oxygen species (ROS). These data indicate that the tetraploids might undergo extensive transcriptome reprogramming of genes involved in ROS scavenging and sugar metabolism, which contributes, synergistically or independently, to the enhanced stress tolerance of the tetraploid. Our results reveal that the tetraploids take priority over the diploid for stress tolerance by maintaining a more robust system of ROS detoxification and osmotic adjustment via elevating antioxidant capacity and sugar accumulation in comparison with the diploid counterpart.

ERF109 of trifoliate orange (Poncirus trifoliata (L.) Raf.) contributes to cold tolerance by directly regulating expression of Prx1 involved in antioxidative process.

Ethylene-responsive factors (ERFs) have been revealed to play essential roles in a variety of physiological and biological processes in higher plants. However, functions and regulatory pathways of most ERFs in cold stress remain largely unclear. Here, we identified PtrERF109 of trifoliate orange (Poncirus trifoliata (L.) Raf.) and deciphered its role in cold tolerance. PtrERF109 was drastically up-regulated by cold, ethylene and dehydration, but repressed by salt. PtrERF109 was localized in the nucleus and displayed transcriptional activity, and the C terminus is required for the activation. Overexpression of PtrERF109 conferred enhanced cold tolerance in transgenic tobacco and lemon plants, whereas VIGS (virus-induced gene silencing)-mediated suppression of PtrERF109 in trifoliate orange led to increased cold susceptibility. PtrERF109 overexpression caused extensive transcriptional reprogramming of several suites of stress-responsive genes. Prx1 encoding class III peroxidase (POD) was one of the antioxidant genes exhibiting the greatest induction. PtrERF109 was shown to directly bind to the promoter of PtrPrx1 (trifoliate orange Prx1 homologue) and positively activated its expression. In addition, the PtrERF109-overexpressing plants exhibited significantly higher POD activity and accumulated dramatically less H2 O2 and were more tolerant to oxidative stress, whereas the VIGS plants exhibited opposite trends, in comparison with wild type. Taken together, these results indicate that PtrERF109 as a positive regulator contributes to imparting cold tolerance by, at least partly, directly regulating the POD-encoding gene to maintain a robust antioxidant capacity for effectively scavenging the reactive oxygen species. Our findings gain insight into better understanding of transcriptional regulation of antioxidant genes in response to cold stress.

Comparative transcriptome analysis of Poncirus trifoliata identifies a core set of genes involved in arbuscular mycorrhizal symbiosis.

The perennial woody plants of citrus are one of the most important fruit crops in the world and largely depends on arbuscular mycorrhizal symbiosis (AMS) to obtain essential nutrients from soil. However, the molecular aspects of AMS in citrus and perennial woody plants in general have largely been understudied. We used RNA-sequencing to identify differentially expressed genes in roots of Poncirus trifoliata upon mycorrhization by the AM fungus Glomus versiforme and evaluated their conservation by comparative transcriptome analyses with four herbaceous model plants. We identified 282 differentially expressed genes in P. trifoliata, including orthologs of 21 genes with characterized roles in AMS and 83 genes that are considered to be conserved in AM-host plants. Comparative transcriptome analysis revealed a 'core set' of 156 genes from P. trifoliata whose orthologous genes from at least three of the five species also exhibited similar transcriptional changes during AMS. Functional analysis of one of these conserved AM-induced genes, a 3-keto-acyl-ACP reductase (FatG) involved in fatty acid biosynthesis, confirmed its involvement in AMS in Medicago truncatula. Our results identify a core transcriptional program for AMS that is largely conserved between P. trifoliata and other plants. The comparative transcriptomics approach adds to previous phylogenomics studies to identify conserved genes required for AMS.

Ameliorative effects of boron on aluminum induced variations of cell wall cellulose and pectin components in trifoliate orange (Poncirus trifoliate (L.) Raf.) rootstock.

Aluminum (Al) phytotoxicity is a major limitation in the production of crops in the soils with pH ≤ 5. Boron (B) is indispensable nutrient for the development of higher plants and B role has been reported in the alleviation Al toxicity. Trifoliate orange rootstock was grown in two B and two Al concentrations. The results of the present study showed that Al toxicity adversely inhibited root elongation and exhibited higher oxidative stress in terms of H2O2 and O2- under B-deficiency. Additionally, the X-ray diffraction (XRD) analysis confirmed the increase of the cellulose crystallinity in the cell wall (CW). Al-induced remarkable variations in the CW components were prominent in terms of alkali-soluble pectin, 2-keto-3-deoxyoctonic acid (KDO) and the degree of methyl-esterification (DME) of pectin. Interesting, B supply reduced the pectin (alkali-soluble) under Al toxicity. Moreover, the results of FTIR (Fourier transform infrared spectroscopy) and 13C-NMR (13C nuclear magnetic resonance) spectra revealed the decrease of carboxyl groups and cellulose by B application during Al exposure. Furthermore, B supply tended to decrease the Al uptake, CW thickness and callose formation. The study concluded that B could mitigate Al phytotoxicity by shielding potential Al binding sites and by reducing Al induced alterations in the CW cellulose and pectin components.

Mycorrhiza stimulates root-hair growth and IAA synthesis and transport in trifoliate orange under drought stress.

Root-hair growth and development regulated by soil microbes is associated with auxin. In this background, we hypothesized that mycorrhizal fungal inoculation induces greater root-hair growth through stimulated auxin synthesis and transport under water stress conditions. Trifoliate orange (Poncirus trifoliata) was inoculated with an arbuscular mycorrhizal (AM) fungus (Funneliformis mosseae) under well-watered (WW) and drought stress (DS) for 9 weeks. Compared with non-AM seedlings, AM seedlings displayed significantly higher density, length, and diameter of root hairs and root indoleacetic acid (IAA) level, whereas lower total root IAA efflux, regardless of soil moisture status. Root PtYUC3 and PtYUC8 involved in IAA biosynthesis were up-regulated by mycorrhization under WW and DS, whereas AM-modulated expression in PtTAA1, PtTAR2, PtYUC4, and PtYUC6 depended on status of soil moisture. Mycorrhizal inoculation down-regulated the transcript level of root auxin efflux carriers like PtPIN1 and PtPIN3, whereas significantly up-regulated the expression of root auxin-species influx carriers like PtABCB19 and PtLAX2 under DS. These results indicated that AMF-stimulated greater root-hair growth of trifoliate orange under DS that is independent on AMF species is related with mycorrhiza-modulated auxin synthesis and transport, which benefits the host plant to enhance drought tolerance.

Molecular cloning and characterization of PtrZPT2-1, a ZPT2 family gene encoding a Cys2/His2-type zinc finger protein from trifoliate orange (Poncirus trifoliata (L.) Raf.) that enhances plant tolerance to multiple abiotic stresses.

In plants, most Cys2/His2 (C2H2) zinc finger proteins with two zinc finger domains (ZPT2) are involved in abiotic stress responses. In this study, a ZPT2 family gene PtrZPT2-1 was cloned from trifoliate orange (Poncirus trifoliata (L.) Raf.). PtrZPT2-1 is composed of 245 amino acids, has a putative molecular weight of 25.99kDa and an isoelectric point of 8.41. PtrZPT2-1 contained two C2H2 zinc finger domains, one nuclear localization signal (B-box), one transcription repression domain (DLN-box), and one protein-protein interaction domain (L-box). PtrZPT2-1 was localized to the nucleus. The PtrZPT2-1 expression was strongly induced by cold, drought, salt and ABA stresses. Overexpression of PtrZPT2-1 increased the survival rates, and the ABA, soluble sugar and proline levels but decreased the ion leakage, the malondialdehyde (MDA) content and reduced the H2O2 accumulation in the transgenic tobacco after cold, drought or salt treatments. Furthermore, the expression levels of 15 abiotic stress-related genes were significantly increased in the transgenic tobacco overexpressing PtrZPT2-1 after cold, drought or salt stress treatments. Our results indicated that overexpression of PtrZPT2-1 in the transgenic tobacco could improve the cold, drought and salt resistance of the plants by increasing the levels of osmotic regulatory solutes and decreasing the accumulation of H2O2.

Alleviation of drought stress by mycorrhizas is related to increased root H2O2 efflux in trifoliate orange.

The Non-invasive Micro-test Technique (NMT) is used to measure dynamic changes of specific ions/molecules non-invasively, but information about hydrogen peroxide (H2O2) fluxes in different classes of roots by mycorrhiza is scarce in terms of NMT. Effects of Funneliformis mosseae on plant growth, H2O2, superoxide radical (O2·-), malondialdehyde (MDA) concentrations, and H2O2 fluxes in the taproot (TR) and lateral roots (LRs) of trifoliate orange seedlings under well-watered (WW) and drought stress (DS) conditions were studied. DS strongly inhibited mycorrhizal colonization in the TR and LRs, whereas mycorrhizal inoculation significantly promoted plant growth and biomass production. H2O2, O2·-, and MDA concentrations in leaves and roots were dramatically lower in mycorrhizal seedlings than in non-mycorrhizal seedlings under DS. Compared with non-mycorrhizal seedlings, mycorrhizal seedlings had relatively higher net root H2O2 effluxes in the TR and LRs especially under WW, as well as significantly higher total root H2O2 effluxes in the TR and LRs under WW and DS. Total root H2O2 effluxes were significantly positively correlated with root colonization but negatively with root H2O2 and MDA concentrations. It suggested that mycorrhizas induces more H2O2 effluxes of the TR and LRs, thus, alleviating oxidative damage of DS in the host plant.

Physiologic, Anatomic, and Gene Expression Changes in Citrus sunki, Poncirus trifoliata, and Their Hybrids After 'Candidatus Liberibacter asiaticus' Infection.

Huanglongbing (HLB) is a destructive disease of citrus caused by phloem-limited bacteria, namely 'Candidatus Liberibacter asiaticus' (Las), 'Candidatus Liberibacter africanus', and 'Candidatus Liberibacter americanus'. Although there are no known HLB-resistant citrus species, studies have reported Poncirus trifoliata as being more tolerant. Assuming that callose deposition in the phloem of infected plants can inhibit translocation of photosynthetic products and cause starch accumulation, we compared callose deposition in petioles and starch accumulation in infected leaves of three genotypes (Citrus sinensis, C. sunki, and P. trifoliata) and 15 hybrids (C. sunki × P. trifoliata). Compared with the mock-inoculated plants, higher bacterial counts and greater accumulation of callose and starch were found in C. sinensis, C. sunki, and 10 of the hybrid plants. Lower titer and fewer metabolic changes due to Las infection were observed in P. trifoliata and in two Las-positive hybrids while three hybrids were Las-negative. Callose accumulation was linked to and correlated with genes involved in phloem functionality and starch accumulation was linked to up-regulation of genes involved in starch biosynthesis and repression of those related to starch breakdown. Lower expression of genes involved in phloem functionality in resistant and tolerant plants can partially explain the absence of distinct disease symptoms associated with starch accumulation that are usually observed in HLB-susceptible genotypes.

A NAC Transcription Factor Represses Putrescine Biosynthesis and Affects Drought Tolerance.

Arginine decarboxylase (ADC)-mediated putrescine biosynthesis plays an important role in plant stress responses, but the transcriptional regulation of ADC in response to abiotic stress is not well understood. We isolated a NAM, ATAF1/2, and CUC (NAC) domain-containing transcription factor, PtrNAC72, from trifoliate orange (Poncirus trifoliata) by yeast one-hybrid screening. PtrNAC72, localized to the nucleus, binds specifically to the promoter of PtADC and acts as a transcriptional repressor. PtrNAC72 expression was induced by cold, drought, and abscisic acid. ADC messenger RNA abundance and putrescine levels were decreased in transgenic tobacco (Nicotiana nudicaulis) plants overexpressing PtrNAC72 but increased, compared with the wild type, in an Arabidopsis (Arabidopsis thaliana) transfer DNA insertion mutant, nac72 While transgenic tobacco lines overexpressing PtrNAC72 were more sensitive to drought, plants of the Arabidopsis nac72 mutant exhibited enhanced drought tolerance, consistent with the accumulation of reactive oxygen species in the tested genotypes. In addition, exogenous application of putrescine to the overexpression lines restored drought tolerance, while treatment with d-arginine, an ADC inhibitor, compromised the drought tolerance of nac72 Taken together, these results demonstrate that PtrNAC72 is a repressor of putrescine biosynthesis and may negatively regulate the drought stress response, at least in part, via the modulation of putrescine-associated reactive oxygen species homeostasis.

The miR396b of Poncirus trifoliata Functions in Cold Tolerance by Regulating ACC Oxidase Gene Expression and Modulating Ethylene-Polyamine Homeostasis.

MicroRNAs (miRNAs) are non-coding regulatory molecules that play important roles in a variety of biological processes. Although a number of cold-responsive miRNAs have been computationally identified, functions and mechanisms of most miRNAs are not well understood. Herein, the function of trifoliate orange [Poncirus trifoliata (L.) Raf.] miRNA396b (ptr-miR396b) in cold tolerance and its potential regulatory module were investigated. Compared with the wild type (WT), transgenic lemon (Citrus limon) plants overexpressing ptr-MIR396b, the precursor of ptr-miR396b, displayed enhanced cold tolerance. Ptr-miR396b was experimentally confirmed to guide the cleavage of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO). The overexpressing lines exhibited a reduction in ACO transcript levels and ethylene content compared with the WT, and the expression pattern of ACO was opposite to that of ptr-miR396b in response to cold stress. In addition, the transgenic lines exhibited higher levels of free polyamines and mRNA abundance of polyamine biosynthetic genes than WT plants under cold treatment, consistent with reduced reactive oxygen species (ROS) accumulation in the former. Taken together, this study demonstrates that ptr-miR396b positively regulates cold tolerance through reducing ACO transcript levels, thereby repressing ethylene synthesis and simultaneously promoting polyamine synthesis, leading to enhanced ROS scavenging. Identification of the ptr-miR396b-ACO regulatory module provides new insights into the molecular mechanism underlying the reduction of ethylene production under cold.

An Integrated View of Whole-Tree Hydraulic Architecture. Does Stomatal or Hydraulic Conductance Determine Whole Tree Transpiration?

Hydraulic conductance exerts a strong influence on many aspects of plant physiology, namely: transpiration, CO2 assimilation, growth, productivity or stress response. However we lack full understanding of the contribution of root or shoot water transport capacity to the total water balance, something which is difficult to study in trees. Here we tested the hypothesis that whole plant hydraulic conductance modulates plant transpiration using two different seedlings of citrus rootstocks, Poncirus trifoliata (L.) Raf. and Cleopatra mandarin (Citrus reshni Hort ex Tan.). The two genotypes presented important differences in their root or shoot hydraulic conductance contribution to whole plant hydraulic conductance but, even so, water balance proved highly dependent on whole plant conductance. Further, we propose there is a possible equilibrium between root and shoot hydraulic conductance, similar to that between shoot and root biomass production, which could be related with xylem anatomy.

Effects of grafting on key photosynthetic enzymes and gene expression in the citrus cultivar Huangguogan.

Grafting influences scion photosynthetic capacity and fruit quality. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which strongly affects photosynthetic rate, and Rubisco activase (RCA), which regulates Rubisco activity, are two key photosynthetic enzymes. However, little information is available regarding the effect of grafting on the concentration and expression of Rubisco and RCA in the citrus cultivar Huangguogan. The objective of this study was to investigate the effect of grafting Huangguogan plants onto trifoliate orange, tangerine, and orange on: 1) the concentration of Rubisco and RCA; 2) the mRNA levels of rbcL, rbcS, and rca; and 3) fruit quality. Overall, the results showed that when Huangguogan plants budded on tangerine and orange, they had better fruit quality, while on trifoliate orange they had higher Rubisco concentration. Tangerine and orange are probably the most suitable rootstocks for Huangguogan plants given the environmental conditions of Sichuan Province, China.

PtrABF of Poncirus trifoliata functions in dehydration tolerance by reducing stomatal density and maintaining reactive oxygen species homeostasis.

Abscisic acid-responsive element (ABRE)-binding factors (ABFs) play important roles in abiotic stress responses; however, the underlying mechanisms are poorly understood. In this study, it is reported that overexpression of Poncirus trifoliata PtrABF significantly enhanced dehydration tolerance. The transgenic lines displayed smaller stomatal apertures, reduced stomatal density/index, and lower expression levels of genes associated with stomatal development. PtrABF was found to interact with PtrICE1, a homologue of ICE1 (Inducer of CBF Expression 1) that has been shown to be critical for stomatal development. Microarray analysis revealed that a total of 70 genes were differentially expressed in the transgenic line, 42 induced and 28 repressed. At least two units of ABREs and coupling elements were present in the promoters of most of the induced genes, among which peroxidase and arginine decarboxylase were verified as bona fide targets of PtrABF. Transgenic plants exhibited higher antioxidant enzyme activities and free polyamine levels, but lower levels of reactive oxygen species (ROS) and malondialdehyde. Polyamines were revealed to be associated with ROS scavenging in the transgenic plants due to a modulation of antioxidant enzymes triggered by signalling mediated by H2O2 derived from polyamine oxidase (PAO)-mediated catabolism. Taken together, the results indicate that PtrABF functions positively in dehydration tolerance by limiting water loss through its influence on stomatal movement or formation and maintaining ROS homeostasis via modulation of antioxidant enzymes and polyamines through transcriptional regulation of relevant target genes.