Leaf Protoplasts Research Articles

Overexpression of Populus transcription factor PtrTALE12 increases axillary shoot development by regulating WUSCHEL expression

The TALE (Three Amino acid Loop Extension) transcription factor family has been shown to control meristem formation and organogenesis in plants. To understand the functional roles of the TALE family in woody perennials, each of the TALE members of Populus trichocarpa was overexpressed in Arabidopsis as a proxy. Among them, the overexpression of PtrTALE12 (i.e., 35S::PtrTALE12) resulted in a dramatic increase of axillary shoot development with early flowering. Interestingly, expression of WUSCHEL (WUS), a central regulator of both apical and axillary meristem formation, was significantly increased in the 35S::PtrTALE12 Arabidopsis plants. Conversely, WUS expression was downregulated in 35S::PtrTALE12-SRDX (short transcriptional repressor domain) plants. Further analysis found that PtrTALE12, expressed preferentially in meristem tissues, directly regulates WUS expression in transient activation assays using Arabidopsis leaf protoplast. Yeast two-hybrid assays showed that PtrTALE12 interacts with SHOOT MERISTEMLESS (STM); however, the interaction does not affect the WUS expression. In addition, expression of both CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) genes was suppressed accordingly for early flowering 35S::PtrTALE12 Arabidopsis. Indeed, transgenic poplars overexpressing PtrTALE12 as well as Arabidopsis plants overexpressing AtBLH11, a close homolog of PtrTALE12, phenocopied the 35S::PtrTALE12 Arabidopsis (i.e., increased axillary shoot development). Taken together, our results suggest that PtrTALE12 functions as a positive regulator of axillary shoot formation in both Arabidopsis and poplar.

The Maize AP2/EREBP Transcription Factor ZmEREB160 Enhances Drought Tolerance in Arabidopsis

Drought is a main factor affecting plant growth and crop production. APETALA2/Ethylene Responsive Element Binding Protein (AP2/EREBP) transcription factors (TFs) are involved in the response to abiotic stress and their functions in ABA signaling and the regulation of drought response have been intensively studied. However, AP2/EREBP TFs have been limited in the maize. The objective of our study was to identify the function of the novel maize AP2/EREBP gene ZmEREB160. Expression levels analysis in maize revealed that ZmEREB160 is significantly induced by PEG6000, NaCl and ABA treatment. ZmEREB160 localized to the nucleus when transiently expressed in Arabidopsis leaf protoplasts. ZmEREB160 activated the reporter gene and exhibited transcriptional activation activity in yeast cells. When overexpression of ZmEREB160 in Arabidopsis significantly enhanced tolerance to osmotic and ABA stress, overexpressed seedlings were longer roots under ABA and mannitol treatments compared with wild type. In addition, overexpression of ZmEREB160 Arabidopsis seedlings was found to elevate survival rate compared with wild type plants under drought stress. During the drought treatment, qRT-PCR assays showed that the expression levels of ABA/drought stress-related genes, ABI2, ABI5, COR15, DREB2A and RD29B were up-regulated in ZmEREB160 transgenic plants, transgenic plants accumulated more proline content than wild type plants. These results indicate that ZmEREB160 functions in response to drought and ABA stresses, and participates in ABA signaling pathway and may enhance drought tolerance.

Ectopic expression of HaNAC1, an ATAF transcription factor from Haloxylon ammodendron, improves growth and drought tolerance in transgenic Arabidopsis

NAC transcription factors play a pivotal role in plant growth, development and response to abiotic stress. However, their biological functions in desert trees are largely unknown. In this work, the NAC transcription factor HaNAC1 from Haloxylon ammodendron, a typical wooden plant normally grown in desert, was isolated, and its possible role in plant growth and resistance to drought stress was investigated. HaNAC1 encodes an ATAF subfamily transcription factor containing one NAC domain with five conserved regions. Quantitative real time PCR analyses revealed that HaNAC1 was ubiquitously expressed in various tissues and organs such as roots, stems, leaves and seeds, with a predominant expression in stems. Further studies demonstrated that expression of HaNAC1 was significantly induced by osmotic stress in Haloxylon ammodendron seedlings, and subcellular localization analysis indicated that GFP-HaNAC1 fusion protein was localized to the nucleus in Arabidopsis leaf protoplast. Ectopic expression of HaNAC1 led to promoted growth and drought tolerance in transgenic Arabidopsis, accompanied with up-regulated expression of stress-inducible marker genes, and increased accumulation of proline, IAA and ABA under both normal and drought stress conditions. In addition, co-immunoprecipitation and Bi-molecular fluorescence complementation assays illustrated that HaNAC1 directly interacted with AtNAC32. All these results suggest that HaNAC1 is involved in both the growth and drought resistance of Haloxylon ammodendron, and could be used as a promising candidate gene for the breeding of crops with augmented tolerance to drought stress.

Membrane-Specific Targeting of Tail-Anchored Proteins SECE1 and SECE2 Within Chloroplasts.

Membrane proteins that are imported into chloroplasts must be accurately targeted in order to maintain the identity and function of the highly differentiated internal membranes. Relatively little is known about the targeting information or pathways that direct proteins with transmembrane domains to either the inner envelope or thylakoids. In this study, we focused on a structurally simple class of membrane proteins, the tail-anchored proteins, which have stroma-exposed amino-terminal domains and a single transmembrane domain within 30 amino acids of the carboxy-terminus. SECE1 and SECE2 are essential tail-anchored proteins that function as components of the dual SEC translocases in chloroplasts. SECE1 localizes to the thylakoids, while SECE2 localizes to the inner envelope. We have used transient expression in Arabidopsis leaf protoplasts and confocal microscopy in combination with a domain-swapping strategy to identify regions that contain important targeting determinants. We show that membrane-specific targeting depends on features of the transmembrane domains and the short C-terminal tails. We probed the contributions of these regions to targeting processes further through site-directed mutagenesis. We show that thylakoid targeting still occurs when changes are made to the tail of SECE1, but changing residues in the tail of SECE2 abolishes inner envelope targeting. Finally, we discuss possible parallels between sorting of tail-anchored proteins in the stroma and in the cytosol.

A cell death assay in barley and wheat protoplasts for identification and validation of matching pathogen AVR effector and plant NLR immune receptors

BackgroundPlant disease resistance to host-adapted pathogens is often mediated by host nucleotide-binding and leucine-rich repeat (NLR) receptors that detect matching pathogen avirulence effectors (AVR) inside plant cells. AVR-triggered NLR activation is typically associated with a rapid host cell death at sites of attempted infection and this response constitutes a widely used surrogate for NLR activation. However, it is challenging to assess this cell death in cereal hosts.ResultsHere we quantify cell death upon NLR-mediated recognition of fungal pathogen AVRs in mesophyll leaf protoplasts of barley and wheat. We provide measurements for the recognition of the fungal AVRs AvrSr50 and AVRa1 by their respective cereal NLRs Sr50 and Mla1 upon overexpression of the AVR and NLR pairs in mesophyll protoplast of both, wheat and barley.ConclusionsOur data demonstrate that the here described approach can be effectively used to detect and quantify death of wheat and barley cells induced by overexpression of NLR and AVR effectors or AVR effector candidate genes from diverse fungal pathogens within 24 h.

ZmDREB1A Regulates RAFFINOSE SYNTHASE Controlling Raffinose Accumulation and Plant Chilling Stress Tolerance in Maize.

Raffinose accumulation is positively correlated with plant chilling stress tolerance; however, the understanding of the function and regulation of raffinose metabolism under chilling stress remains in its infancy. RAFFINOSE SYNTHASE (RAFS) is the key enzyme for raffinose biosynthesis. In this study, we report that two independent maize (Zea mays) zmrafs mutant lines, in which raffinose was completely abolished, were more sensitive to chilling stress and their net photosynthetic product (total soluble sugars and starch) accumulation was significantly decreased compared with controls after chilling stress. A similar characterization of the maize dehydration responsive element (DRE)-binding protein 1A mutant (zmdreb1a) showed that ZmRAFS expression and raffinose content were significantly decreased compared with its control under chilling stress. Overexpression of maize ZmDREB1A in maize leaf protoplasts increased ZmDREB1A amounts, which consequently upregulated the expression of maize ZmRAFS and the Renilla LUCIFERASE (Rluc), which was controlled by the ZmRAFS promoter. Deletion of the single dehydration-responsive element (DRE) in the ZmRAFS promoter abolished ZmDREB1A's influence on Rluc expression, while addition of three copies of the DRE in the ZmRAFS promoter dramatically increased Rluc expression when ZmDREB1A was simultaneously overexpressed. Electrophoretic mobility shift assays and chromatin immunoprecipitation-quantitative PCR demonstrated that ZmDREB1A directly binds to the DRE motif in the promoter of ZmRAFS both in vitro and in vivo. These data demonstrate that ZmRAFS, which was directly regulated by ZmDREB1A, enhances both raffinose biosynthesis and plant chilling stress tolerance.

Tyrosine Kinase-Dependent Defense Responses Against Herbivory in Arabidopsis.

Tyrosine (Tyr) phosphorylation (TP) is important for promotion of plants’ signaling. Arabidopsis calcium-dependent protein kinase related protein kinases (CRK2 and CRK3) phosphorylate Tyr residues of a subset of transcription factors (TFs), including herbivory-responsive ethylene response factor 13 (ERF13), but the in vivo functions of these kinases in plant defense responses and development remain to be clarified. We show that when CRKs were coexpressed with ERF13 in Arabidopsis leaf protoplasts, the transcription activity regulated via ERF13 was elevated by CRK2 but not CRK3 or their kinase-dead form mutants. Moreover, this elevation was abolished when a Tyr-phosphorylation mutant of ERF was coexpressed with CRK2, indicating that CRK2 serves as an effector of ERF13 mediated by Tyr-phosphorylation. Moreover, CRK2 and CRK3 acted as effectors of RAP2.6 and WRKY14, respectively. CRK-overexpressing lines and knockout mutants of Arabidopsis plants showed increased and decreased expression levels of the defensin gene PDF1.2 in leaves, respectively, conferring on the plants modulated defense properties against the generalist herbivore Spodoptera litura. However, these lines did not show any obvious developmental defects, indicating that CRKs play a role in defense responses but not in the ordinary growth or development of plants. Transcription of both CRK2 and CRK3 was positively regulated by jasmonate signaling and abscisic acid (ABA) signaling upon herbivory. Our findings suggest that these phytohormone-responsive CRKs work coordinately for plant defense responses via Tyr phosphorylation of herbivory-responsive regulators.

PdMYB118, isolated from a red leaf mutant of Populus deltoids, is a new transcription factor regulating anthocyanin biosynthesis in poplar

A new anthocyanin biosynthesis transcription factor PdMYB118, which could be used for the genetic engineering of colorful tree species, was indentified from a red leaf mutant of Populus deltoids. In higher plants, the biosynthesis of anthocyanins is regulated by several classes of transcription factors (TFs), including R2R3-MYB, bHLH and WD-repeat proteins. In this work, we isolated an MYB gene regulating anthocyanin biosynthesis from a red leaf mutant of Populus deltoids, which accumulated more anthocyanins in the leaves and showed higher expression levels of anthocyanin biosynthesis genes than did the wild type. Gene expression analyses of all TFs regulating anthocyanin biosynthesis demonstrated that only a MYB118 homologous gene, PdMYB118, was up-regulated in the mutant compared with the wide type. Subcellular localization analyses in poplar leaf mesophyll protoplasts showed that PdMYB118-YFP fusion protein was specifically located in nucleus. When transiently expressed in poplar leaf protoplasts, PdMYB118 specifically promoted the expression of anthocyanidin biosynthesis genes. Dual-luciferase assays revealed that PdMYB118 can directly activate the promoters of these genes. When overexpressed in Shanxin Yang (P. davidiana × P. bolleana), a hybrid clone commercially grown for landscaping in the northern part of China, transgenic plants overexpressing PdMYB118 produced more anthocyanins in the leaves and turned their color into redness when grown in both greenhouse and field. Consistently, transcripts of some important anthocyanidin biosynthesis genes were significantly increased in the leaves of transgenic plants. All these results indicate that PdMYB118 functions as an essential transcription factor regulating anthocyanin biosynthesis in poplar and could be used for the genetic engineering of colorful tree species.

Identification and characterization of a novel stay-green QTL that increases yield in maize.

SummaryFunctional stay‐green is a valuable trait that extends the photosynthetic period, increases source capacity and biomass and ultimately translates to higher grain yield. Selection for higher yields has increased stay‐green in modern maize hybrids. Here, we report a novel QTL controlling functional stay‐green that was discovered in a mapping population derived from the Illinois High Protein 1 (IHP1) and Illinois Low Protein 1 (ILP1) lines, which show very different rates of leaf senescence. This QTL was mapped to a single gene containing a NAC‐domain transcription factor that we named nac7. Transgenic maize lines where nac7 was down‐regulated by RNAi showed delayed senescence and increased both biomass and nitrogen accumulation in vegetative tissues, demonstrating NAC7 functions as a negative regulator of the stay‐green trait. More importantly, crosses between nac7 RNAi parents and two different elite inbred testers produced hybrids with prolonged stay‐green and increased grain yield by an average 0.29 megagram/hectare (4.6 bushel/acre), in 2 years of multi‐environment field trials. Subsequent RNAseq experiments, one employing nac7 RNAi leaves and the other using leaf protoplasts overexpressing Nac7, revealed an important role for NAC7 in regulating genes in photosynthesis, chlorophyll degradation and protein turnover pathways that each contribute to the functional stay‐green phenotype. We further determined the putative target of NAC7 and provided a logical extension for the role of NAC7 in regulating resource allocation from vegetative source to reproductive sink tissues. Collectively, our findings make a compelling case for NAC7 as a target for improving functional stay‐green and yields in maize and other crops.

Plastidial and Mitochondrial Malonyl CoA-ACP Malonyltransferase is Essential for Cell Division and Its Overexpression Increases Storage Oil Content.

Malonyl-acyl carrier protein (ACP) is a key building block for the synthesis of fatty acids, which are important components of cell membranes, storage oils and lipid-signaling molecules. Malonyl CoA-ACP malonyltransferase (MCAMT) catalyzes the production of malonyl-ACP and CoA from malonyl-CoA and ACP. Here, we report that MCAMT plays a critical role in cell division and has the potential to increase the storage oil content in Arabidopsis. The quantitative real-time PCR and MCAMT promoter:GUS analyses showed that MCAMT is predominantly expressed in shoot and root apical meristems, leaf hydathodes and developing embryos. The fluorescent signals of MCAMT:eYFP were observed in both chloroplasts and mitochondria of tobacco leaf protoplasts. In particular, the N-terminal region (amino acid residues 1-30) of MCAMT was required for mitochondrial targeting. The Arabidopsis mcamt-1 and -2 mutants exhibited an embryo-lethal phenotype because of the arrest of embryo development at the globular stage. The transgenic Arabidopsis expressing antisense MCAMT RNA showed growth retardation caused by the defects in cell division. The overexpression of MCAMT driven by the promoter of the senescence-associated 1 (SEN1) gene, which is predominantly expressed in developing seeds, increased the seed yield and storage oil content of Arabidopsis. Taken together, the plastidial and mitochondrial MCAMT is essential for Arabidopsis cell division and is a novel genetic resource useful for enhancing storage oil content in oilseed crops.

The PILNCR1-miR399 Regulatory Module Is Important for Low Phosphate Tolerance in Maize.

The regulation of adaptive responses to phosphorus (P) deficiency by the microRNA399 (miR399)/PHOSPHATE2 (PHO2) pathway has been well studied in Arabidopsis (Arabidopsis thaliana) but not in maize (Zea mays). Here, we show that miR399 transcripts are strongly induced in maize by phosphate (Pi) deficiency. Transgenic maize plants that overexpressed MIR399b accumulated excessive amounts of P in their shoots and displayed typical Pi-toxicity phenotypes. We reannotated ZmPHO2 with an additional 1,165 bp of the 5' untranslated region. miR399-guided posttranscriptional repression of ZmPHO2 was mainly observed in the P-efficient lines. We identified Pi-deficiency-induced long-noncoding RNA1 (PILNCR1) from our strand-specific RNA libraries. Transient expression assays in Nicotiana benthamiana and maize leaf protoplasts demonstrated that PILNCR1 inhibits ZmmiR399-guided cleavage of ZmPHO2 The abundance of PILNCR1 was significantly higher in P-inefficient lines than in P-efficient lines, which is consistent with the abundance of ZmmiR399 transcripts. These results indicate that the interaction between PILNCR1 and miR399 is important for tolerance to low Pi in maize.

Decoding the Divergent Subcellular Location of Two Highly Similar Paralogous LEA Proteins.

Many mitochondrial proteins are synthesized as precursors in the cytosol with an N-terminal mitochondrial targeting sequence (MTS) which is cleaved off upon import. Although much is known about import mechanisms and MTS structural features, the variability of MTS still hampers robust sub-cellular software predictions. Here, we took advantage of two paralogous late embryogenesis abundant proteins (LEA) from Arabidopsis with different subcellular locations to investigate structural determinants of mitochondrial import and gain insight into the evolution of the LEA genes. LEA38 and LEA2 are short proteins of the LEA_3 family, which are very similar along their whole sequence, but LEA38 is targeted to mitochondria while LEA2 is cytosolic. Differences in the N-terminal protein sequences were used to generate a series of mutated LEA2 which were expressed as GFP-fusion proteins in leaf protoplasts. By combining three types of mutation (substitution, charge inversion, and segment replacement), we were able to redirect the mutated LEA2 to mitochondria. Analysis of the effect of the mutations and determination of the LEA38 MTS cleavage site highlighted important structural features within and beyond the MTS. Overall, these results provide an explanation for the likely loss of mitochondrial location after duplication of the ancestral gene.

Melatonin biosynthesis enzymes recruit WRKY transcription factors to regulate melatonin accumulation and transcriptional activity on W-box in cassava.

Melatonin is widely involved in growth, development, and stress responses in plants. Although the melatonin synthesis enzymes have been identified in various plants, their interacting proteins remain unknown. Herein, overexpression of tryptophan decarboxylase 2 (MeTDC2)-interacting proteins, N-acetylserotonin O-methyltransferase 2 (MeASMT2) interacting proteins, and N-acetylserotonin O-methyltransferase 3 (MeASMT3) in cassava leaf protoplasts resulted in more melatonin than when other enzymes were overexpressed. Through yeast two-hybrid, 14 MeTDC2-interacting proteins, 24 MeASMT2 interacting proteins, and 9 MeASMT3-interacting proteins were identified. Notably, we highlighted MeWRKY20 and MeWRKY75 as common interacting proteins of the 3 enzymes, as evidenced by yeast two-hybrid, and in vivo bimolecular fluorescence complementation (BiFC). Moreover, co-overexpression of MeTDC2/MeASMT2/3 with MeWRKY20/75 in cassava leaf protoplasts did not only activated the transcriptional activities of MeWRKY20 and MeWRKY75 on W-box, but also induced the effects of MeTDC2, MeASMT2/3 on endogenous melatonin levels. Taken together, 3 melatonin synthesis enzymes (MeTDC2, MeASMT2/3) interact with MeWRKY20/75 to form a protein complex in cassava. This information significantly extends the knowledge of the complex modulation of plant melatonin signaling.

Expression of TpNRAMP5, a metal transporter from Polish wheat (Triticum polonicum L.), enhances the accumulation of Cd, Co and Mn in transgenic Arabidopsis plants.

TpRNAMP5 is mainly expressed in the plasma membrane of roots and basal stems. It functions as a metal transporter for Cd, Mn and Co accumulation. Numerous natural resistance-associated macrophage proteins (NRAMPs) have been functionally identified in various plant species, including Arabidopsis, rice, soybean and tobacco, but no information is available on NRAMP genes in wheat. In this study, we isolated a TpNRAMP5 from dwarf Polish wheat (DPW, Triticum polonicum L.), a species with high tolerance to Cd and Zn. Expression pattern analysis revealed that TpNRAMP5 is mainly expressed in roots and basal stems of DPW. TpNRAMP5 was localized at the plasma membrane of Arabidopsis leaf protoplast. Expression of TpNRAMP5 in yeast significantly increased yeast sensitivity to Cd and Co, but not Zn, and enhanced Cd and Co concentrations. Expression of TpNRAMP5 in Arabidopsis significantly increased Cd, Co and Mn concentrations in roots, shoots and whole plants, but had no effect on Fe and Zn concentrations. These results indicate that TpNRAMP5 is a metal transporter enhancing the accumulation of Cd, Co and Mn, but not Zn and Fe. Genetic manipulation of TpNRAMP5 can be applied inthe future to limit the transfer of Cd from soil to wheat grains, thereby protecting human health.

Two G-box-like elements essential to high gene expression of SlAKR4B in tomato leaves.

Aldo-keto reductases (AKRs) play important roles in aldehyde detoxification as well as primary and secondary metabolism in plants. We previously reported inducible expression of a Solanum lycopersicum AKR4B (SlAKR4B) in tomato leaves treated with salicylic acid and jasmonic acid, and high promoter activity of SlAKR4B in tomato leaf protoplasts. In this study, we investigated the expression response of SlAKR4B in the tomato leaves with infiltration treatment and the cis-element(s) involved in high promoter activity. Gene expression analysis in tomato leaf protoplasts and buffer-infiltrated tomato leaves suggested that cell damage caused the increased expression of SlAKR4B. Promoter activity of SlAKR4B was significantly reduced by mutation of two G-box like elements. It is suggested that the two G-box like elements are responsible for the high promoter activity.

Investigation of the Phaseolus vulgaris circadian clock and the repressive role of the PvTOC1 factor by a newly established in vitro system

The circadian clock is crucial for the synchronization of an organism’s physiology and metabolism with the geophysical time. In plants, previous work on the common bean (Phaseolus vulgaris) has identified various differing aspects of clock function compared to the widely studied Arabidopsis thaliana clock. However, transformation of legumes for the study of the circadian clock regulatory mechanisms is extremely laborious. In the present work, we describe an easy-to-follow and rapid method of preparing bean leaf protoplasts with high transformation potential and a functional circadian clock. In this system, we show that application of trichostatin A differentially changes the expression levels of several clock genes. More importantly, we investigate the effect of the clock protein PvTOC1 (Phaseolus vulgaris TIMING OF CAB EXPRESSION 1) on the activity of bean circadian promoters. We present new evidence on the function of PvTOC1 as a repressor of the promoter activity of its own gene, mediated by its conserved CCT (CONSTANS, CO-LIKE and TOC1) domain. Using our protoplast system we were able to uncover functions of the bean circadian clock and to identify an additional target of the PvTOC1clock transcription factor, not previously reported.

RAV transcription factors are essential for disease resistance against cassava bacterial blight via activation of melatonin biosynthesis genes.

With 1 AP2 domain and 1 B3 domain, 7 MeRAVs in apetala2/ethylene response factor (AP2/ERF) gene family have been identified in cassava. However, the in vivo roles of these remain unknown. Gene expression assays showed that the transcripts of MeRAVs were commonly regulated after Xanthomonas axonopodis pv manihotis (Xam) and MeRAVs were specifically located in plant cell nuclei. Through virus-induced gene silencing (VIGS) in cassava, we found that MeRAV1 and MeRAV2 are essential for plant disease resistance against cassava bacterial blight, as shown by the bacterial propagation of Xam in plant leaves. Through VIGS in cassava leaves and overexpression in cassava leave protoplasts, we found that MeRAV1 and MeRAV2 positively regulated melatonin biosynthesis genes and the endogenous melatonin level. Further investigation showed that MeRAV1 and MeRAV2 are direct transcriptional activators of 3 melatonin biosynthesis genes in cassava, as evidenced by chromatin immunoprecipitation-PCR in cassava leaf protoplasts and electrophoretic mobility shift assay. Moreover, cassava melatonin biosynthesis genes also positively regulated plant disease resistance. Taken together, this study identified MeRAV1 and MeRAV2 as common and upstream transcription factors of melatonin synthesis genes in cassava and revealed a model of MeRAV1 and MeRAV2-melatonin biosynthesis genes-melatonin level in plant disease resistance against cassava bacterial blight.

RMR (Receptor Membrane RING-H2) type 1 and 2 show different promoter activities and subcellular localizations in Arabidopsis thaliana

Soluble vacuolar proteins reach their compartments of final accumulation through the binding with specific transmembrane cargo receptors. In Arabidopsis thaliana two different families of receptors have been characterized. The AtVSRs (Vacuolar Sorting Receptor), which are known to be involved in the protein sorting to lytic vacuoles (LV), and the AtRMRs (Receptor Membrane RING-H2), for which there is less evidence for a role in the traffic to the protein storage vacuole (PSV). In this study we investigated the localization and tissue expression of two RMRs (AtRMR1 and 2) in their species of origin, A. thaliana. Our experiments using leaf protoplasts and transgenic plants supported previous results of subcellular localization in Nicotiana benthamiana that visualized AtRMR1 and 2 in the cisternae of endoplasmic reticulum (ER) and in the trans-Golgi network (TGN), respectively. The promoter activities of AtRMR1 and AtRMR2 detected in transgenic A. thaliana lines suggest that the expression of these two receptors only partially overlap in some organs and tissues. These results suggest that AtRMR1 and 2 are not functionally redundant, but could also interact and participate in the same cellular process in tissues with an overlapping expression.

Two MATE Transporters with Different Subcellular Localization are Involved in Al Tolerance in Buckwheat.

Buckwheat (Fagopyrum esculentum) shows high tolerance to aluminum (Al) toxicity, but the molecular mechanisms responsible for this high Al tolerance are still poorly understood. Here, we investigated the involvement of two MATE (multi-drug and toxic compound extrusion) genes in Al tolerance. Both FeMATE1 and FeMATE2 showed efflux transport activity for citrate, but not for oxalate when expressed in Xenopus oocytes. A transient assay with buckwheat leaf protoplasts using green fluorescent protein (GFP) fusion showed that FeMATE1 was mainly localized to the plasma membrane, whereas FeMATE2 was localized to the trans-Golgi and Golgi. The expression of FeMATE1 was induced by Al only in the roots, but that of FeMATE2 was up-regulated in both the roots and leaves. Furthermore, the expression of both genes only responded to Al toxicity, but not to other stresses including low pH, cadmium (Cd) and lanthanum (La). Heterologous expression of FeMATE1 or FeMATE2 in the Arabidopsis mutant atmate partially rescued its Al tolerance. Expression of FeMATE1 also partially recovered the Al-induced secretion of citrate in the transgenic lines, whereas expression of FeMATE2 did not complement the citrate secretion. Further physiological analysis showed that buckwheat roots also secreted citrate in addition to oxalate in response to Al in a dose-responsive manner. Taken together, our results indicate that FeMATE1 is involved in the Al-activated citrate secretion in the roots, while FeMATE2 is probably responsible for transporting citrate into the Golgi system for the internal detoxification of Al in the roots and leaves of buckwheat.

Overexpression of a PIP1 Gene from Salicornia bigelovii in Tobacco Plants Improves Their Drought Tolerance

Aquaporin (AQP) proteins can transport water and other small molecules through cellular membranes and are one of the first targets of stress-induced signaling in plants. A number of AQP genes have been identified from glycophytes, and their functions have been studied. However, the reports on AQPs from halophytes and their precise role in abiotic stress response are still rare. In this study, we have identified a PIP1 subgroup AQP gene, designated SbPIP1, from the euhalophyte Salicornia bigelovii and characterized it by overexpressing in tobacco plants. SbPIP1 transcript was induced by cold, but suppressed by NaCl and polyethylene glycol (PEG). Transient expression of GFP (green fluorescent protein)-SbPIP fusion protein indicated its localization in the plasma membrane. Overexpression of SbPIP1 in tobacco (Nicotiana tabacum) plants increased their drought tolerance. Leaf protoplasts from transgenic tobacco plants absorbed water more quickly than those from wild type (WT) plants when they were put into hypotonic solution. In addition, the transgenic tobacco plants possessed higher relative water content (RWC) and proline content, but lower levels of malondialdehyde (MDA) and less ion leakage (IL) when compared with WT under the treatment of the different concentrations of PEG. Taken together, our results demonstrate that heterologous expression of SbPIP1 in tobacco plants confers them drought stress tolerance by reducing membrane injury and increasing the ability to retain water.