NaCl-induced CsRCI2E and CsRCI2F interact with aquaporin CsPIP2;1 to reduce water transport in Camelina sativa L.

Rare cold-inducible 2 (RCI2) proteins are small hydrophobic proteins that are known to be localized in cellular membranes. The function of RCI2 proteins has been reported to be associated with low-temperature, salt, and drought stress tolerances as a membrane potential regulator; however, the specific functions are still unknown. The PIP2 (plasma membrane intrinsic protein 2) aquaporins are proteins that transport water and small solutes into the cell. The expression and activity of PIP2 proteins, like RCI2, are also related to salt- and drought-stress tolerance. In this study, we identified novel protein interactions between RCI2 and PIP2; 1, including protein accumulation changes in the bioenergy crop Camelina sativa L. under various NaCl stress conditions. Accumulation of both CsRCI2E and CsRCI2F proteins increased with NaCl stress; however, to differing levels depending on the NaCl stress intensity. A co-immunoprecipitation test revealed interaction between CsRCI2E-CsPIP2 and CsRCI2F-CsPIP2. Moreover, co-expression of the four CsRCI2 proteins with CsPIP2; 1 in Xenopus laevis oocytes reduced water transport activity. Furthermore, the abundance of CsPIP2; 1 protein was decreased under CsRCI2E and CsRCI2F co-expression. These results suggest that NaCl-induced expression of CsRCI2E and CsRCI2F contributes to the regulation of CsPIP2; 1.

Changes in fatty acid content and composition between wild type and CsHMA3 overexpressing Camelina sativa under heavy-metal stress.

KEY MESSAGE: Under heavy-metal stress, CsHMA3 overexpressing transgenic Camelina plants displayed not only a better quality, but also a higher quantity of unsaturated fatty acids in their seeds compared with wild type. Camelina sativa L. belongs to the Brassicaceae family and is frequently used as a natural vegetable oil source, as its seeds contain a high content of fatty acids. In this study, we observed that, when subjected to heavy metals (Cd, Co, Zn and Pb), the seeds of CsHMA3 (Heavy-Metal P1B-ATPase 3) transgenic lines retained their original golden yellow color and smooth outline, unlike wild-type seeds. Furthermore, we investigated the fatty acids content and composition of wild type and CsHMA3 transgenic lines after heavy metal treatments compared to the control. The results showed higher total fatty acid amounts in seeds of CsHMA3 transgenic lines compared with those in wild-type seeds under heavy-metal stresses. In addition, the compositions of unsaturated fatty acids-especially 18:1 (oleic acid), 18:2 (linoleic acid; only in case of Co treatment), 18:3 (linolenic acid) and 20:1 (eicosenoic acid)-in CsHMA3 overexpressing transgenic lines treated with heavy metals were higher than those of wild-type seeds under the same conditions. Furthermore, reactive oxygen species (ROS) contents in wild-type leaves and roots when treated with heavy metal were higher than in CsHMA3 overexpressing transgenic lines. These results indicate that overexpression of CsHMA3 affects fatty acid composition and content-factors that are responsible for the fuel properties of biodiesel-and can alleviate ROS accumulation caused by heavy-metal stresses in Camelina. Due to these factors, we propose that CsHMA3 transgenic Camelina can be used for phytoremediation of metal-contaminated soil as well as for oil production.

Development of Haploid Plants by Shed-Microspore Culture in Platycodon grandiflorum (Jacq.) A. DC.

Anther and microspore cultures are efficient methods for inducing haploids in plants. The microspore culture by chromosome-doubling method can produce double haploid lines, developing pure lines within the first or second generations. This study aimed to induce haploid plants in Platycodon grandiflorum using the shed-microspore culture method. P. grandiflorum floral buds (n = 1503) were cultured in six types of medium to induce haploids. Anthers were placed on a solid-liquid double-layer medium and cold pre-treated at 9 °C for one week, followed by incubation in the dark at 25 °C. Embryogenesis was observed after approximately 70 days of culture, producing haploid plants through regeneration. Of the 1503 floral buds, embryos developed in 120 buds, resulting in the induction of 402 individuals. Among the media used, Schenk and Hildebrandt (SH) and 1/2SH exhibited high efficiency, with embryogenesis ratios of 12% and 13.4%, respectively. Additionally, the highest embryogenesis ratio (15.3%) was observed in flower buds sized 10 mm or less. Therefore, we established shed-microspore culture conditions to induce haploids in P. grandiflorum. Using this method, haploids can be efficiently induced in P. grandiflorum, shortening the breeding period by enabling the rapid development of inbred lines.

Overexpression of PIP2;5 aquaporin alleviates effects of low root temperature on cell hydraulic conductivity and growth in Arabidopsis.

The effects of low root temperature on growth and root cell water transport were compared between wild-type Arabidopsis (Arabidopsis thaliana) and plants overexpressing plasma membrane intrinsic protein 1;4 (PIP1;4) and PIP2;5. Descending root temperature from 25°C to 10°C quickly reduced cell hydraulic conductivity (L(p)) in wild-type plants but did not affect L(p) in plants overexpressing PIP1;4 and PIP2;5. Similarly, when the roots of wild-type plants were exposed to 10°C for 1 d, L(p) was lower compared with 25°C. However, there was no effect of low root temperature on L(p) in PIP1;4- and PIP2;5-overexpressing plants after 1 d of treatment. When the roots were exposed to 10°C for 5 d, L(p) was reduced in wild-type plants and in plants overexpressing PIP1;4, whereas there was still no effect in PIP2;5-overexpressing plants. These results suggest that the gating mechanism in PIP1;4 may be more sensitive to prolonged low temperature compared with PIP2;5. The reduction of L(p) at 10°C in roots of wild-type plants was partly restored to the preexposure level by 5 mm Ca(NO(3))(2) and protein phosphatase inhibitors (75 nm okadaic acid or 1 µm Na(3)VO(4)), suggesting that aquaporin phosphorylation/dephosphorylation processes were involved in this response. The temperature sensitivity of cell water transport in roots was reflected by a reduction in shoot and root growth rates in the wild-type and PIP1;4-overexpressing plants exposed to 10°C root temperature for 5 d. However, low root temperature had no effect on growth in plants overexpressing PIP2;5. These results provide strong evidence for a link between growth at low root temperature and aquaporin-mediated root water transport in Arabidopsis.

Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation.

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.

Zn tolerance of novel Colocasia esculenta metallothionein and its domains in Escherichia coli and tobacco.

Contrary to extensive researches on the roles of metallothioneins (MTs) in metal tolerance of animals, the roles of plant MTs in metal tolerance are largely under investigation. In this study, we evaluated the functional role of type 2 MT from Colocasia esculenta (CeMT2b) in Zn tolerance of tobacco and E. coli cells. Under Zn-stress conditions, transgenic tobacco overexpressing CeMT2b displayed much better seedling growth, a significant decrease in the levels of H(2)O(2) and an increase in Zn accumulation compared with the wild type. Overexpression of CeMT2b in E. coli greatly enhanced Zn tolerance and Zn accumulation under Zn stresses compared with control cells. CeMT2b bound 5.38 ± 0.29 atoms of Zn per protein. To identify a structural domain of CeMT2b for Zn binding, we investigated the growth of E. coli expressing each of the N-terminal, C-terminal, and central linker domains or a CNC motif deletion from the C-terminus of full-length CeMT2b. The results showed that the CNC motif is required for Zn tolerance, and the N-terminal domain is more effective in Zn tolerance than the C-terminal domain. Taken together, our results provide direct evidence for functional contributions of CeMT2b in Zn tolerance of tobacco and E. coli cells.

CsRCI2D enhances high-temperature stress tolerance in Camelina sativa L. through endo-membrane trafficking from the plasma membrane.

Rare Cold Inducible 2s (RCI2s) are hydrophobic proteins in cell membranes that participate in abiotic stress tolerance mechanisms. Additionally, they are used as traceable membrane trafficking markers in endocytosis studies. Plants regulate cell homeostasis through endocytosis by limiting the activity of plasma membrane transporter proteins to adapt to stressful conditions. In this study, we found high temperature (HT) stress-induced membrane trafficking of RCI2D in Camelina sativa L. The gene expression and protein synthesis were increased by HT stress at 37 °C. Moreover, rapid membrane trafficking of CsRCI2D was traced by multiple-phase membrane fractionation using sucrose density gradients and compared with CsRCI2E/F/G from the same protein family subgroup. The distribution of CsRCI2s was shown to be similar to that of the clathrin heavy chain, which is known as a major endocytosis protein. Subcellular localization of CsRCI2D was observed in the plasma membrane and endo-membranes and overlapped with membrane lipids. CsRCI2D co-localized with lipids, and its overexpression increased the intracellular lipid content compared to that of wild-type camelina. Moreover, transgenic camelina lines showed enhanced HT stress tolerance during germination and hypocotyl elongation when compared to the wild type. These results suggest that HT-induced CsRCI2D membrane trafficking enhances HT stress tolerance in camelina.

The R-R-type MYB-like transcription factor, AtMYBL, is involved in promoting leaf senescence and modulates an abiotic stress response in Arabidopsis.

Functional analysis of a putative novel transcription factor Arabidopsis MYB-like protein designated AtMYBL, which contains two predicted DNA-binding domains, was performed. The physiological role of the R-R-type MYB-like transcription factor has not been reported in any plant. Analyses of an AtMYBL promoter-ß-glucuronidase (GUS) construct revealed substantial gene expression in old leaves and induction of GUS activity by ABA and salt stress. AtMYBL-overexpressing plants displayed a markedly enhanced leaf senescence phenotype. Moreover, the ectopic expression of the AtMYBL gene was very significantly influential in senescence parameters including Chl content, membrane ion leakage and the expression of senescence-related genes. Although the seed germination rate was improved under ABA and saline stress conditions in the AtMYBL-overexpressing plants, decreased salt tolerance was evident compared with the wild type and atmybl RNA interference lines during later seedling growth when exposed to long-term salt stress, indicating that AtMYBL protein is able to developmentally regulate stress sensitivity. Furthermore, AtMYBL protein activated the transcription of a reporter gene in yeast. Green fluorescent protein-tagged AtMYBL was localized in the nuclei of transgenic Arabidopsis cells. Taken together, these results suggest that AtMYBL functions in the leaf senescence process, with the abiotic stress response implicated as a putative potential transcription factor.

Increasing Monounsaturated Fatty Acid Contents in Hexaploid Camelina sativa Seed Oil by FAD2 Gene Knockout Using CRISPR-Cas9.

Seed oils are used as edible oils and increasingly also for industrial applications. Although high-oleic seed oil is preferred for industrial use, most seed oil is high in polyunsaturated fatty acids (PUFAs) and low in monounsaturated fatty acids (MUFAs) such as oleic acid. Oil from Camelina, an emerging oilseed crop with a high seed oil content and resistance to environmental stress, contains 60% PUFAs and 30% MUFAs. Hexaploid Camelina carries three homoeologs of FAD2, encoding fatty acid desaturase 2 (FAD2), which is responsible for the synthesis of linoleic acid from oleic acid. In this study, to increase the MUFA contents of Camelina seed oil, we generated CsFAD2 knockout plants via CRISPR-Cas9-mediated gene editing using the pRedU6fad2EcCas9 vector containing DsRed as a selection marker, the U6 promoter to drive a single guide RNA (sgRNA) covering the common region of the three CsFAD2 homoeologs, and an egg-cell-specific promoter to drive Cas9 expression. We analyzed CsFAD2 homoeolog-specific sequences by PCR using genomic DNA from transformed Camelina leaves. Knockout of all three pairs of FAD2 homoeologs led to a stunted bushy phenotype, but greatly enhanced MUFA levels (by 80%) in seeds. However, transformants with two pairs of CsFAD2 homoeologs knocked out but the other pair wild-type heterozygous showed normal growth and a seed MUFAs production increased up to 60%. These results provide a basis for the metabolic engineering of genes that affect growth in polyploid crops through genome editing.

Arabidopsis thaliana Rubisco small subunit transit peptide increases the accumulation of Thermotoga maritima endoglucanase Cel5A in chloroplasts of transgenic tobacco plants.

Over the past decade various approaches have been used to increase the expression level of recombinant proteins in plants. One successful approach has been to target proteins to specific subcellular sites/compartments of plant cells, such as the chloroplast. In the study reported here, hyperthermostable endoglucanase Cel5A was targeted into the chloroplasts of tobacco plants via the N-terminal transit peptide of nuclear-encoded plastid proteins. The expression levels of Cel5A transgenic lines were then determined using three distinct transit peptides, namely, the light-harvesting chlorophyll a/b-binding protein (CAB), Rubisco small subunit (RS), and Rubisco activase (RA). RS:Cel5A transgenic lines produced highly stable active enzymes, and the protein accumulation of these transgenic lines was up to 5.2% of the total soluble protein in the crude leaf extract, remaining stable throughout the life cycle of the tobacco plant. Transmission election microscopy analysis showed that efficient targeting of Cel5A protein was under the control of the transit peptide.

Subcellular Journey of Rare Cold Inducible 2 Protein in Plant Under Stressful Condition.

Rare cold inducible 2 (RCI2) proteins are small hydrophobic membrane proteins in plants, and it has been widely reported that RCI2 expressions are dramatically induced by salt, cold, and drought stresses in many species. The RCI2 proteins have been shown to regulate plasma membrane (PM) potential and enhance abiotic stress tolerance when over-expressed in plants. RCI2 protein structures contain two transmembrane domains that are thought to be PM intrinsic proteins and have been observed at the PM and endomembranes. However, cellular trafficking of RCI2s are not fully understood. In this review, we discussed (i) general properties of RCI2s characterized in many species, (ii) the uses of RCI2s as a tracer in live cell imaging analyses and when they are fused to fluorescence proteins during investigations into vesicle trafficking, and (iii) RCI2 functionalities such as their involvement in rapid diffusion, endocytosis, and protein interactions. Consequently, the connection between physiological characteristics of RCI2s and traffic of RCI2s interacting membrane proteins might be helpful to understand role of RCI2s contributing abiotic stresses tolerance.

Development of a Rapid Selection System for Salt-Resistant Mutants of Nicotiana benthamiana through Protoplast Culture after Gamma Irradiation.

We aimed to develop a novel technology capable of rapidly selecting mutant plant cell lines. Salt resistance was chosen as a rapid selection trait that is easily applicable to protoplast-derived cell colonies. Mesophyll protoplasts were cultured in a medium supplemented with 0, 50, 100, 150, 200, 250, and 300 mM NaCl. At NaCl concentrations ≥ 100 mM, cell colony formation was strongly inhibited after 4 weeks of culture. Tobacco protoplasts irradiated with 0, 50, 100, 200, and 400 Gy were then cultured to investigate the effects of radiation intensity on cell division. The optimal radiation intensity was 50 Gy. To develop salt-resistant tobacco mutant plants, protoplasts irradiated with 50 Gy were cultured in a medium containing 100 mM NaCl. The efficiency of cell colony formation from these protoplasts was approximately 0.002%. A salt-resistant mutant callus was selected and proliferated in the same medium and then transferred to a shoot inducing medium for adventitious shoot formation. The obtained shoots were then cultured in a medium supplemented with 200 mM NaCl and developed into normal plantlets. This rapid selection technology for generating salt-resistant tobacco mutants will be useful for the development of crop varieties resistant to environmental stresses.

Isolation and functional characterization of the Arabidopsis salt-tolerance 32 (AtSAT32) gene associated with salt tolerance and ABA signaling.

Recently, we have isolated salt-tolerance genes (SATs) on the basis of the overexpression screening of yeast with a maize cDNA library from kernels. One of the selected genes [salt-tolerance 32 (SAT32)] appears to be a key determinant for salt stress tolerance in yeast cells. Maize SAT32 cDNA encodes for a 49-kDa protein, which is 41% identity with the Arabidopsis salt-tolerance 32 (AtSAT32) unknown gene. Arabidopsis Transfer-DNA (T-DNA) knockout AtSAT32 (atsat32) altered root elongation, including reduced silique length and reduced seed number. In an effort to further assess salinity tolerance in Arabidopsis, we have functionally characterized the AtSAT32 gene and determined that salinity and the plant hormone ABA induced the expression of AtSAT32. The atsat32 mutant was more sensitive to salinity than the wild-type plant. On the contrary, Arabidopsis overexpressing AtSAT32 (35S::AtSAT32) showed enhanced salt tolerance and increased activity of vacuolar H(+)-pyrophosphatase (V-PPase, EC 3.6.1.1) under high-salt conditions. Consistent with these observations, 35S::AtSAT32 plants exhibited increased expression of salt-responsive and ABA-responsive genes, including the Rd29A, Erd15, Rd29B, Rd22 and RAB18 genes. Therefore, our results indicate that AtSAT32 is involved in both salinity tolerance and ABA signaling as a positive regulator in Arabidopsis.

Overexpression of phytochelatin synthase AtPCS2 enhances salt tolerance in Arabidopsis thaliana.

Phytochelatin synthase (PCS) is an enzyme that synthesizes phytochelatins, which are metal-binding peptides. Despite the important role of PCS in heavy metal detoxification or tolerance, the functional role of PCS with respect to other abiotic stresses remains largely unknown. In this study, we determined the function of Arabidopsis thaliana phytochelatin synthase 2 (AtPCS2) in the salt stress response. Expression of AtPCS2 was significantly increased in response to 100 and 200 mM NaCl treatment. AtPCS2-overexpressing transgenic Arabidopsis and tobacco plants displayed increased seed germination rates and seedling growth under high salt stress. In addition, transgenic Arabidopsis subjected to salt stress exhibited enhanced proline accumulation and reduced Na+/K+ ratios compared to wild type plants. Furthermore, decreased levels of hydrogen peroxide (H2O2) and lipid peroxidation were observed in transgenic Arabidopsis compared to wild type specimens. Salt stress greatly reduced transcript levels of CuSOD2, FeSOD2, CAT2, and GR2 in wild type but not transgenic Arabidopsis. Notably, levels of CAT3 in transgenic Arabidopsis were markedly increased upon salt stress, suggesting that low accumulation of H2O2 in transgenic Arabidopsis is partially achieved through induction of CAT. Collectively, these results suggest that AtPCS2 plays a positive role in seed germination and seedling growth under salt stress through a series of indirect effects that are likely involved in H2O2 scavenging, regulation of osmotic adjustment and ion homeostasis.

Inhibition of growth and development of root border cells in wheat by Al.

The production and development of border cells vary with genotype, and they are released in wheat at an earlier stage of root development than other species studied so far. No significant difference was observed in the maximum number of border cells between Al-tolerant (Atlas 66) and Al-sensitive (Scout 66) cultivars in the absence of Al treatment. Al seriously inhibited the production and release of border cells, resulting in clumping of border cells in Scout 66, but less clustering in Atlas 66. The number of border cells released from roots treated with Al is significantly less than that from roots grown without Al treatment. Al treatment induced the death of detached border cells in vitro and they were killed by a 20-h treatment with 25 micro m Al. No significant difference in survival percentage of detached border cells was observed between Atlas 66 and Scout 66, regardless of the presence or absence of Al. The removal of border cells from root tips of both Atlas 66 and Scout 66 enhanced the Al-induced inhibition of root elongation concomitant with increased Al accumulation in the root. These results suggest that border cells adhered to the root tips play a potential role in the protection of root from Al injury in wheat.

Exposure of roots of cucumber (Cucumis sativus) to low temperature severely reduces root pressure, hydraulic conductivity and active transport of nutrients.

When root temperature dropped below 25 degrees C, there was a sharp drop in the root pressure (P(r)) and hydraulic conductivity of excised roots (Lp(r)) of young cucumber (Cucumis sativus L.) seedlings as measured with the root pressure probe. A detailed analysis of root hydraulics provided evidence for a larger reduction in the osmotic component of Lp(r) (77%) in comparison with the hydrostatic component (34%) in response to the exposure of the root system to 13 degrees C. The activity of the plasma membrane H(+)-ATPase (EC 3.6.1.35) was reduced from 30 to 16 micro mol Pi mg(-1) protein h(-1) upon exposure to 8 degrees C for 1 day. Ultrastructural observations showed no evidence of loosening of the microstructure of endodermal cell walls in low temperature (LT)-treated roots. It is concluded that the rapid drop in the P(r) in response to LT is largely caused by a reduction in the activity of the plasma membrane H(+)-ATPase rather than by loosening of the endodermal wall which would cause substantial solute losses. On the other hand, water permeability of root cell membrane at LT was related to changes in the activity (open/closed state) of water channels.

Alteration of leaf shape, improved metal tolerance, and productivity of seed by overexpression of CsHMA3 in Camelina sativa.

BACKGROUND: Camelina sativa (L.) Crantz, known by such popular names as "gold-of-pleasure" and "false flax," is an alternative oilseed crop for biofuel production and can be grown in harsh environments. Considerable interest is now being given to the new concept of the development of a fusion plant which can be used as a soil remediation plant for ground contaminated by heavy metals as well as a bioenergy crop. However, knowledge of the transport processes for heavy metals across Camelina plant membranes is still rudimentary. RESULTS: Firstly, to investigate whether Camelina HMA (heavy metal P1B-ATPase) genes could be used in such a plant, we analyzed the expression patterns of eight HMA genes in Camelina (taken from the root, leaf, stem, flower, and silique). CsHMA3 genes were expressed in all organs. In addition, CsHMA3 was induced in roots and leaves especially after Pb treatment. Heterogeneous expression of CsHMA3 complemented the Pb- or Zn-sensitive phenotype of Δycf1 or Δzrc1 yeast mutant strains. Subsequently, we cloned and overexpressed CsHMA3 in Camelina. The root growth of transgenic lines was better than that in the wild-type plant under heavy metal stress (for Cd, Pb, and Zn). In particular, the transgenic lines showed enhanced Pb tolerance in a wide range of Pb concentrations. Furthermore, the Pb and Zn content in the shoots of the transgenic lines were higher than those in the wild-type plant. These results suggest that overexpression of CsHMA3 might enhance Pb and Zn tolerance and translocation. Also, the transgenic lines displayed a wider leaf shape compared with the wild-type plant due to an induction of genes related to leaf width growth and showed a greater total seed yield compared to the wild type under heavy metal stress. CONCLUSIONS: Our data obtained from physiological and functional analyses using CsHMA3 overexpression plants will be useful to develop a multifunctional plant that can improve the productivity of a bioenergy crop and simultaneously be used to purify an area contaminated by various heavy metals.

The Camelina aquaporin CsPIP2;1 is regulated by phosphorylation at Ser273, but not at Ser277, of the C-terminus and is involved in salt- and drought-stress responses.

Aquaporin (AQP) proteins are involved in water homeostasis in cells at all taxonomic levels of life. Phosphorylation of some AQPs has been proposed to regulate water permeability via gating of the channel itself. We analyzed plasma membrane intrinsic proteins (PIP) from Camelina and characterized their biological functions under both stressful and favorable conditions. A three-dimensional theoretical model of the Camelina AQP proteins was built by homology modeling which could prove useful in further functional characterization of AQPs. CsPIP2;1 was strongly and constitutively expressed in roots and leaves of Camelina, suggesting that this gene is related to maintenance of homeostasis during salt and drought stresses. CsPIP2s exhibited water channel activity in Xenopus oocytes. We then examined the roles of CsPIP2;1 phosphorylation at Ser273 and Ser277 in the regulation of water permeability using phosphorylation mutants. A single deletion strain of CsPIP2;1 was generated to serve as the primary host for testing AQP expression constructs. A Ser277 to alanine mutation (to prevent phosphorylation) did not change CsPIP2;1 water permeability while a Ser273 mutation to alanine did affect water permeability. Furthermore, a CsPIP2;1 point mutation when ectopically expressed in yeast resulted in lower growth in salt and drought conditions compared with controls, and confirmation of Ser273 as the phosphorylation site. Our results support the idea that post-translational modifications in the Ser273 regulatory domains of the C-terminus fine tune water flux through CsPIP2;1.

Molecular cloning, characterization, and stress-responsive expression of genes encoding glycine-rich RNA-binding proteins in Camelina sativa L.

Camelina sativa L. is an oil-seed crop that has potential for biofuel applications. Although the importance of C. sativa as a biofuel crop has increased in recent years, reports demonstrating the stress responsiveness of C. sativa and characterizing the genes involved in stress response of C. sativa have never been published. Here, we isolated and characterized three genes encoding glycine-rich RNA-binding proteins (GRPs) from camelina: CsGRP2a, CsGRP2b, and CsGRP2c. The three CsGRP2 proteins were very similar in amino acid sequence and contained a well-conserved RNA-recognition motif at the N-terminal region and glycine-rich domain at the C-terminal region. To understand the functional roles of CsGRP2s under stress conditions, we investigated the expression patterns of CsGRP2s under various environmental stress conditions. The expressions of the three CsGRP2s were highly up-regulated under cold stress. The expression of CsGRP2a was up-regulated under salt or dehydration stress, whereas the transcript levels of CsGRP2b and CsGRP2c were decreased under salt or dehydration stress conditions. The three CsGRP2s had the ability to complement cold-sensitive Escherichia coli mutants at low temperatures and harbored transcription anti-termination and nucleic acid-melting activities, indicating that the CsGRP2s possess RNA chaperone activity. The CsGRP2a protein was localized to both the nucleus and the cytoplasm. Expression of CsGRP2a in cold-sensitive Arabidopsis grp7 mutant plants resulted in decreased electrolyte leakage at freezing temperatures. Collectively, these results suggest that the stress-responsive CsGRP2s play a role as an RNA chaperone during the stress adaptation process in camelina.

Two aquaporins of Jatropha are regulated differentially during drought stress and subsequent recovery.

Jatropha has potential to be an important bio-fuel crop due to such advantages as high seed oil content and the ability to grow well on marginal lands less suited for food crops. Despite its ability to grow on marginal land, Jatropha is still susceptible to high salt and drought stresses, which can significantly reduce plant growth, stomatal conductance, sap-flow rate, and plant sap volume. This study was undertaken to collect basic knowledge of the physiological and molecular aspects of Jatropha response to salt and drought stresses, and to elucidate how Jatropha recovers from stress. From these studies we identified candidate genes that may be useful for the development of Jatropha cultivars that will grow efficiently in arid and barren lands. Of particular interest, two plasma membrane intrinsic proteins were identified: Jatropha plasma membrane intrinsic protein 1 (JcPIP1) and Jatropha plasma membrane intrinsic protein 2 (JcPIP2). The expression levels of JcPIP1 were dramatically increased in roots, stems, and leaves during the recovery from stress, whereas the JcPIP2 gene transcripts levels were induced in roots and stems during the water deficit stress. The protein levels of JcPIP1 and JcPIP2 were consistent with the gene expression patterns. Based on these results, we hypothesized that JcPIP1 plays a role in the recovery events from water stresses, while JcPIP2 is important in early responses to water stress. Virus induced gene silencing technology revealed that both JcPIP1 and JcPIP2 have positive roles in response to water deficit stresses, but have antagonistic functions at the recovery stage. We suggest that both JcPIP1 and JcPIP2 may play important roles in responses to water deficit conditions and both have potential as targets for genetic engineering.