Chlorotic Phenotype Research Articles

Protein NirP1 regulates nitrite reductase and nitrite excretion in cyanobacteria

When the supply of inorganic carbon is limiting, photosynthetic cyanobacteria excrete nitrite, a toxic intermediate in the ammonia assimilation pathway from nitrate. It has been hypothesized that the excreted nitrite represents excess nitrogen that cannot be further assimilated due to the missing carbon, but the underlying molecular mechanisms are unclear. Here, we identified a protein that interacts with nitrite reductase, regulates nitrogen metabolism and promotes nitrite excretion. The protein, which we named NirP1, is encoded by an unannotated gene that is upregulated under low carbon conditions and controlled by transcription factor NtcA, a central regulator of nitrogen homeostasis. Ectopic overexpression of nirP1 in Synechocystis sp. PCC 6803 resulted in a chlorotic phenotype, delayed growth, severe changes in amino acid pools, and nitrite excretion. Coimmunoprecipitation experiments indicated that NirP1 interacts with nitrite reductase, a central enzyme in the assimilation of ammonia from nitrate/nitrite. Our results reveal that NirP1 is widely conserved in cyanobacteria and plays a crucial role in the coordination of C/N primary metabolism by targeting nitrite reductase.

Loss of function mutations at the tomato SSI2 locus impair plant growth and development by altering the fatty acid desaturation pathway.

The stearoyl-ACP desaturase (SACPD) is a key enzyme in the regulation of saturated to unsaturated fatty acid ratio, playing a crucial role in regulating membrane stability and fluidity, as well as photosynthesis efficiency, which makes it an important research focus in crop species. This study reports the characterization and molecular cloning of pale dwarf (pad), a new tomato (Solanum lycopersicum L.) T-DNA recessive mutant, which exhibits a dwarf and chlorotic phenotype. Functional studies of the T-DNA tagged gene were conducted, including phylogenetic analysis, expression and metabolomic analyses, and generation of CRISPR/Cas9 knockout lines. The cloning of T-DNA flanking genomic sequences and a co-segregation analysis found the pad phenotype was caused by a T-DNA insertion disrupting the tomato homologue of the Arabidopsis SUPPRESSOR OF SALICYLIC ACID INSENSITIVITY 2 (SlSSI2), encoding a plastid localized isoform of SACPD. The phenotype of CRISPR/Cas9 SlSSI2 knockout lines confirmed that the morphological abnormalities in pad plants were due to SlSSI2 loss of function. Functional, metabolomic and expression analyses proved that SlSSI2 disruption causes deficiencies in 18:1 fatty acid desaturation and leads to diminished jasmonic acid (JA) content and increased salicylic acid (SA) levels. Overall, these results proved that SSI2 plays a crucial role in the regulation of polyunsaturated fatty acid profiles in tomato, and revealed that SlSSI2 loss of function results in an inhibited JA-responsive signalling pathway and a constitutively activated SA-mediated defence signalling response. This study lays the foundation for further research on tomato SACPDs and their role in plant performance and fitness.

Chloroplast Damage and Photosynthetic System Disorder Induced Chlorosis in the Leaves of Rice Seedlings under Excessive Biuret

Excessive biuret in fertilizer causes leaf albinism in direct-seeded rice fields. This study aimed to provide a comprehensive understanding of the underlying physiology and molecular mechanisms of leaf chlorosis via biuret using morphophysiological and transcriptome analyses. The induction of biuret in albino rice leaves was examined in a net-growing cultivation bed. Some key morphophysiological indices were measured including biuret content, blade ultrastructure, chlorophyll content, and chlorophyll fluorescence parameters. Candidate genes in the chlorotic leaves under biuret stress were also excavated using transcriptome analysis. Furthermore, physiological and biochemical analyses of the changes in enzyme activities and intermediate metabolite contents in relation to the phenotypic changes in the leaves were carried out. The chlorotic leaves of rice seedlings showed higher biuret accumulation, and the leaves suffered severe damage with higher malondialdehyde contents and low chlorophyll contents. Abnormal chloroplast ultrastructures and thylakoid membrane structure loss were observed in chlorotic leaves under biuret exposure. The related genes involved in the chloroplast development, photosynthesis (including antenna proteins), and carbon fixation pathways were significantly downregulated, which suggests that photosynthesis was destroyed in the chlorotic leaves of rice seedlings. Biuret disturbed the photosynthetic system in chloroplast thylakoid membranes by inhibiting chloroplast development, thereby promoting the formation of the chlorotic leaf phenotype in rice seedlings. Our results promote the understanding of the molecular mechanism of rice in response to biuret toxicity.

Transcriptome-wide m6A methylation in natural yellow leaf of Catalpa fargesii.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA, and involved in various biological processes in plants. However, the distribution features and functions of mRNA m6A methylation have been poorly explored in woody perennial plants. In this study, a new natural variety with yellow-green leaves, named Maiyuanjinqiu, was screened from the seedlings of Catalpa fargesii. Based on the preliminary experiment, the m6A methylation levels in the leaves of Maiyuanjinqiu were significantly higher than those in C. fargesii. Furthermore, a parallel analysis of m6A-seq and RNA-seq was carried out in different leaf color sectors. The result showed that m6A modification were mostly identified around the 3'-untranslated regions (3'-UTR), which was slightly negatively correlated with the mRNA abundance. KEGG and GO analyses showed that m6A methylation genes were associated with photosynthesis, pigments biosynthesis and metabolism, oxidation-reduction and response to stress, etc. The overall increase of m6A methylation levels in yellow-green leaves might be associated with the decreased the expression of RNA demethylase gene CfALKBH5. The silencing of CfALKBH5 caused a chlorotic phenotype and increased m6A methylation level, which further confirmed our hypothesis. Our results suggested that mRNA m6A methylation could be considered as a vital epigenomic mark and contribute to the natural variations in plants.

Overexpression of maize ZmLOX6 in Arabidopsis thaliana enhances damage-induced pentyl leaf volatile emissions that affect plant growth and interaction with aphids.

Pentyl leafy volatiles (PLV) are C5 volatiles produced from polyunsaturated fatty acids by plant 13-lipoxygenases (13-LOX) in concert with other lipid metabolizing enzymes. Unlike related C6 volatiles (GLV, green leafy volatiles), little is known about the biosynthesis and physiological function of PLV in plants. Zea mays LOX6 (ZmLOX6) is an unusual plant LOX that lacks lipid oxygenation activity but acts as a hydroperoxide lyase hypothesized to be specifically involved in PLV synthesis. We overexpressed ZmLOX6 in Arabidopsis thaliana and established that it indeed produces PLVs. Overexpression of ZmLOX6 caused a mild chlorotic phenotype, and induced a similar phenotype in untransformed Col-0 plants grown in close proximity, suggesting that airborne signals, such as PLVs, are responsible for the phenotype. PLV production, dependency on the substrate from endogenous 13-LOX(s), and likely competition with endogenous 13-oxylipin pathway were consistent with the model that ZmLOX6 functions as a hydroperoxide lyase. The abundance of individual PLVs was differentially affected by ZmLOX6 overexpression, and the new profile indicated that ZmLOX6 had reaction products distinct from endogenous PLV-producing activities in the Arabidopsis host plants. ZmLOX6 overexpression also induced a new hormonal status, which is likely responsible for increased attraction and propagation of aphids, nonetheless improving host plant tolerance to aphid infestation.

Genomic Analysis and Delineation of the Tan Spot Susceptibility Locus Tsc1 in Wheat.

The necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) causes the foliar disease tan spot in both bread wheat and durum wheat. Wheat lines carrying the tan spot susceptibility gene Tsc1 are sensitive to the Ptr-produced necrotrophic effector (NE) Ptr ToxC. A compatible interaction results in leaf chlorosis, reducing yield by decreasing the photosynthetic area of leaves. Developing genetically resistant cultivars will effectively reduce disease incidence. Toward that goal, the production of chlorosis in response to inoculation with Ptr ToxC-producing isolates was mapped in two low-resolution biparental populations derived from LMPG-6 × PI 626573 (LP) and Louise × Penawawa (LouPen). In total, 58 genetic markers were developed and mapped, delineating the Tsc1 candidate gene region to a 1.4 centiMorgan (cM) genetic interval spanning 184 kb on the short arm of chromosome 1A. A total of nine candidate genes were identified in the Chinese Spring reference genome, seven with protein domains characteristic of resistance genes. Mapping of the chlorotic phenotype, development of genetic markers, both for genetic mapping and marker-assisted selection (MAS), and the identification of Tsc1 candidate genes provide a foundation for map-based cloning of Tsc1.

The Intron Retention Variant CsClpP3m Is Involved in Leaf Chlorosis in Some Tea Cultivars.

Tea products made from chlorotic or albino leaves are very popular for their unique flavor. Probing into the molecular mechanisms underlying the chlorotic leaf phenotype is required to better understand the formation of these tea cultivars and aid in future practical breeding. In this study, transcriptional alterations of multiple subunit genes of the caseinolytic protease complex (Clp) in the chlorotic tea cultivar ‘Yu-Jin-Xiang’ (YJX) were found. Cultivar YJX possessed the intron retention variant of ClpP3, named as CsClpP3m, in addition to the non-mutated ClpP3. The mutated variant results in a truncated protein containing only 166 amino acid residues and lacks the catalytic triad S182-H206-D255. Quantitative analysis of two CsClpP3 variants in different leaves with varying degrees of chlorosis in YJX and analyses of different chlorotic tea cultivars revealed that the transcript ratios of CsClpP3m over CsClpP3 were negatively correlated with leaf chlorophyll contents. The chlorotic young leaf phenotype was also generated in the transgenic tobacco by suppressing ClpP3 using the RNAi method; complementation with non-mutated CsClpP3 rescued the wild-type phenotype, whereas CsClpP3m failed to complement. Taken together, CsClpP3m is involved in leaf chlorosis in YJX and some other tea cultivars in a dose-dependent manner, likely resulting from the failure of Clp complex assembly due to the truncated sequence of CsClpP3m. Our data shed light on the mechanisms controlling leaf chlorosis in tea plants.

Repairing TALEN-mediated double-strand break by microhomology-mediated recombination in tobacco plastids generates abundant subgenomic DNA

Transcription activator-like effector nuclease (TALEN) technology has been widely used to edit nuclear genomes in plants but rarely for editing organellar genomes. In addition, ciprofloxacin, commonly used to cause the double-strand break of organellar DNA for studying the repair mechanism in plants, confers no organellar selectivity and site-specificity. To demonstrate the feasibility of TALEN-mediated chloroplast DNA editing and to use it for studying the repair mechanism in plastids, we developed a TALEN-mediated editing technology fused with chloroplast transit peptide (cpTALEN) to site-specifically edit the rpoB gene via Agrobacteria-mediated transformation of tobacco leaf. Transgenic plants showed various degrees of chlorotic phenotype. Repairing damaged plastid DNA resulted in point mutation, large deletion and small inversion surrounding the rpoB gene by homologous recombination and/or microhomology-mediated recombination. In an albino line, microhomology-mediated recombination via a pair of 12-bp direct repeats between rpoC2 and ycf2 genes generated the chimeric ycf2-rpoC2 subgenome, with the level about 3- to 5-fold higher for subgenomic DNA than ycf2. Additionally, the expression of chimeric ycf2-rpoC2 transcripts versus ycf2 mRNA agreed well with the level of corresponding DNA. The ycf2-rpoC2 subgenomic DNA might independently and preferentially replicate in plastids.

Growth, agronomic characteristics and nutritional attributes of sorghum (Sorghum bicolor) genotypes resistant to ALS inhibitor herbicides

AbstractResistance to acetolactate synthase (ALS) inhibitor herbicides discovered in wild relatives of sorghum was introgressed into cultivated types, but many of the derived lines display interveinal chlorosis at seedling stages. The objectives of this study were to examine the effect of chlorotic phenotype on growth and crop performance with and without herbicide application. Thirty‐six chlorotic and normal ALS‐resistant lines and susceptible check were evaluated in two experimental sets using randomized complete block design in split‐plot arrangement over a period of three years in four replications. Data were collected on leaf chlorophyll content, chlorophyll fluorescence, plant height, biomass and agronomic traits (days to anthesis, plant height and grain yield and yield components). Grain protein, starch, fat and ash were estimated using near‐infrared spectroscopy. The data showed interveinal chlorosis has significantly delayed flowering, but both chlorosis and herbicide treatment did not have negative effects on final yield and nutritional profile. The study suggests ALS‐resistant sorghums with minimal yellowing effect can be developed, and the trait can be commercially deployed without drawback on yield and utilization attributes.

An Argon-Ion-Induced Pale Green Mutant of Arabidopsis Exhibiting Rapid Disassembly of Mesophyll Chloroplast Grana.

Argon-ion beam is an effective mutagen capable of inducing a variety of mutation types. In this study, an argon ion-induced pale green mutant of Arabidopsis thaliana was isolated and characterized. The mutant, designated Ar50-33-pg1, exhibited moderate defects of growth and greening and exhibited rapid chlorosis in photosynthetic tissues. Fluorescence microscopy confirmed that mesophyll chloroplasts underwent substantial shrinkage during the chlorotic process. Genetic and whole-genome resequencing analyses revealed that Ar50-33-pg1 contained a large 940 kb deletion in chromosome V that encompassed more than 100 annotated genes, including 41 protein-coding genes such as TYRAAt1/TyrA1, EGY1, and MBD12. One of the deleted genes, EGY1, for a thylakoid membrane-localized metalloprotease, was the major contributory gene responsible for the pale mutant phenotype. Both an egy1 mutant and F1 progeny of an Ar50-33-pg1 × egy1 cross-exhibited chlorotic phenotypes similar to those of Ar50-33-pg1. Furthermore, ultrastructural analysis of mesophyll cells revealed that Ar50-33-pg1 and egy1 initially developed wild type-like chloroplasts, but these were rapidly disassembled, resulting in thylakoid disorganization and fragmentation, as well as plastoglobule accumulation, as terminal phenotypes. Together, these data support the utility of heavy-ion mutagenesis for plant genetic analysis and highlight the importance of EGY1 in the structural maintenance of grana in mesophyll chloroplasts.

FASCICLIN-LIKE 18 Is a New Player Regulating Root Elongation in Arabidopsis thaliana.

The plasticity of root development represents a key trait that enables plants to adapt to diverse environmental cues. The pattern of cell wall deposition, alongside other parameters, affects the extent, and direction of root growth. In this study, we report that FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 18 (FLA18) plays a role during root elongation in Arabidopsis thaliana. Using root-specific co-expression analysis, we identified FLA18 to be co-expressed with a sub-set of genes required for root elongation. FLA18 encodes for a putative extra-cellular arabinogalactan protein from the FLA-gene family. Two independent T-DNA insertion lines, named fla18-1 and fla18-2, display short and swollen lateral roots (LRs) when grown on sensitizing condition of high-sucrose containing medium. Unlike fla4/salt overly sensitive 5 (sos5), previously shown to display short and swollen primary root (PR) and LRs under these conditions, the PR of the fla18 mutants is slightly longer compared to the wild-type. Overexpression of the FLA18 CDS complemented the fla18 root phenotype. Genetic interaction between either of the fla18 alleles and sos5 reveals a more severe perturbation of anisotropic growth in both PR and LRs, as compared to the single mutants and the wild-type under restrictive conditions of high sucrose or high-salt containing medium. Additionally, under salt-stress conditions, fla18sos5 had a small, chlorotic shoot phenotype, that was not observed in any of the single mutants or the wild type. As previously shown for sos5, the fla18-1 and fla18-1sos5 root-elongation phenotype is suppressed by abscisic acid (ABA) and display hypersensitivity to the ABA synthesis inhibitor, Fluridon. Last, similar to other cell wall mutants, fla18 root elongation is hypersensitive to the cellulose synthase inhibitor, Isoxaben. Altogether, the presented data assign a new role for FLA18 in the regulation of root elongation. Future studies of the unique vs. redundant roles of FLA proteins during root elongation is anticipated to shed a new light on the regulation of root architecture during plant adaptation to different growth conditions.

Ectopic maltase alleviates dwarf phenotype and improves plant frost tolerance of maltose transporter mutants.

Maltose, the major product of starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves, exits the chloroplast via the maltose exporter1 MEX1. Consequently, mex1 loss-of-function plants exhibit substantial maltose accumulation, a starch-excess phenotype and a specific chlorotic phenotype during leaf development. Here, we investigated whether the introduction of an alternative metabolic route could suppress the marked developmental defects typical for mex1 loss-of-function mutants. To this end, we ectopically expressed in mex1 chloroplasts a functional maltase (MAL) from baker's yeast (Saccharomyces cerevisiae, chloroplastidial MAL [cpMAL] mutants). Remarkably, the stromal MAL activity substantially alleviates most phenotypic peculiarities typical for mex1 plants. However, the cpMAL lines contained only slightly less maltose than parental mex1 plants and their starch levels were, surprisingly, even higher. These findings point to a threshold level of maltose responsible for the marked developmental defects in mex1. While growth and flowering time were only slightly retarded, cpMAL lines exhibited a substantially improved frost tolerance, when compared to wild-types. In summary, these results demonstrate the possibility to bypass the MEX1 transporter, allow us to differentiate between possible starch-excess and maltose-excess responses, and demonstrate that stromal maltose accumulation prevents frost defects. The latter insight may be instrumental for the development of crop plants with improved frost tolerance.

CFM1, a member of the CRM-domain protein family, functions in chloroplast group II intron splicing in Setaria viridis.

The chloroplast RNA splicing and ribosome maturation (CRM) domain is a RNA-binding domain found in a plant-specific protein family whose characterized members play essential roles in splicing group I and group II introns in mitochondria and chloroplasts. Together, these proteins are required for splicing of the majority of the approximately 20 chloroplast introns in land plants. Here, we provide evidence from Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. A Setaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs: trnI, trnV and trnA. Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole-plant chlorotic phenotypes. Co-immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in the cfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes.

The OsABCI7 Transporter Interacts with OsHCF222 to Stabilize the Thylakoid Membrane in Rice.

The thylakoid membrane is a highly complex membrane system in plants and plays crucial roles in the biogenesis of the photosynthetic apparatus and plant development. However, the genetic factors involved in chloroplast development and its relationship with intracellular metabolites are largely unknown. Here, a rice (Oryza sativa) chlorotic and necrotic leaf1 (cnl1) mutant was identified and map-based cloning revealed that a single base substitution followed by a 6-bp deletion in the ATP-binding cassette transporter I family member7 (OsABCI7) resulted in chlorotic and necrotic leaves with thylakoid membrane degradation, chlorophyll breakdown, photosynthesis impairment, and cell death in cnl1 Furthermore, the expression of OsABCI7 was inducible under lower temperatures, which severely affected cnl1 chloroplast development, and etiolated cnl1 seedlings were unable to recover to a normal green state under light conditions. Functional complementation and overexpression showed that OsABCI7 could rescue the cnl1 chlorotic and necrotic phenotype. OsABCI7 interacted with HIGH CHLOROPHYLL FLUORESCENCE222 (OsHCF222) to regulate cellular reactive oxygen species (ROS) homeostasis for thylakoid membrane stability. OsABCI7 localized to thylakoid membranes, while OsHCF222 targeted to endoplasmic reticulum and chloroplasts. Exogenous application of ascorbic acid eased the yellowish leaf phenotype by increasing chlorophyll content and alleviating ROS stress in cnl1 Unlike cnl1, the CRISPR/Cas9-mediated OsHCF222 knockout lines showed chlorotic leaves but were seedling lethal. Our results provide insight into the functions of ABC transporters in rice, especially within the relationship between ROS homeostasis and stability of thylakoid membranes.

Importance of non-systemic leaf autophagy for suppression of zinc starvation induced-chlorosis

ABSTRACTAutophagy, which is one of the self-degradation systems, promotes intracellular zinc (Zn) recycling under Zn deficiency (−Zn) in plants. Therefore, autophagy defective plants show severe chlorosis under −Zn. Root is the plant organ which directly exposed to Zn deficient environment, however, in our recent study, −Zn symptom was prominently observed in leaves as chlorosis. Here, we conducted micrografting to determine which organ’s autophagic activities are important to suppress the −Zn induced chlorosis. Grafted plants that have autophagic activities only in roots or leaves were grown under −Zn and then compared chlorotic phenotypes among them. As a result, regardless of the autophagic activities in rootstocks, −Zn induced-chlorosis in leaves was occurred only when autophagy in scion was defective. This data indicates that Zn resupplied by autophagic degradation in root cells could not contribute to suppress the chlorosis in leaves. Thus, autophagy in the aerial part is critical for controlling −Zn induced-chlorosis in leaves. Taken together, along with our recently reported data, we conclude that the mechanism of Zn resupply by autophagic degradation is not systemic throughout the plant but rather a local system.

Functional Analysis of <i>Tadhn1312</i> by Virus-Induced Gene Silencing (VIGS) in Common Wheat (<i>Triticum aestivum</i> L.)

Virus-induced gene silencing (VIGS) is an important tool for gene function analysis in plants. In the present study, the function identification of a candidate gene named Tadhn1312 encoding Dehydrin protein obtained from the high-throughput transcriptome was carried out by VIGS. Leaves appeared the chlorotic phenotype and the transcript level of TaPDS decreased rapidly at 7 days after the inoculation by BSMV: TaPDS, which indicated that the virus had successfully infected wheat leaves and the BSMV system was efficient. The chlorophyll content of leaves with BSMV: TaPDS and BSMV: Tadhn1312 inoculation decreased at 7 days, and reached significant level compared with the control. After the inoculation by BSMV: Tadhn1312, the transcript level of Tadhn1312 was rapidly decreased at 7 days, and reached the minimum value at 21 days, indicated that Tadhn1312 had been silenced. The spike differentiation procession of wheat plants inoculated by BSMV: Tadhn1312 was late than that inoculated by BSMV: 00. The result showed that the silencing of Tadhn1312 prolonged the spike differentiation process, illustrating that Tadhn1312 gene was involved in the spike differentiation process of wheat directly or indirectly.

Combining isotopically non-stationary metabolic flux analysis with proteomics to unravel the regulation of the Calvin-Benson-Bassham cycle in Synechocystis sp. PCC 6803

Photosynthetic microorganisms are increasingly being investigated as a sustainable alternative to existing bio-industrial processes, converting CO2 into desirable end products without the use of carbohydrate feedstock. The Calvin-Benson-Bassham (CBB) cycle is the main pathway of carbon fixation metabolism in photosynthetic organisms. In this study, we analyzed the metabolic fluxes in two strains of the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) that overexpressed fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) and transketolase (TK), respectively. These two potential carbon flux control enzymes in the CBB cycle had previously been shown to improve biomass accumulation when overexpressed under air and low light (15 μmol m−2 s−1) conditions (Liang and Lindblad, 2016). We measured the growth rates of Synechocystis under atmospheric and high (3% v/v) CO2 conditions at 80 μmol m−2 s−1. Surprisingly, the cells overexpressing transketolase (tktA) demonstrated no significant increase in growth rates when CO2 was increased, suggesting an altered carbon flux distribution and a potential metabolic bottleneck in carbon fixation. Moreover, the tktA strain had an increased susceptibility to oxidative stress under high light as revealed by its chlorotic phenotype under high light conditions. In contrast, the fructose-1,6/sedoheptulose-1,7-bisphosphatase (70glpX) and wild-type cells demonstrated increases in growth rates as expected.To investigate the disparate phenotypical responses of these different Synechocystis strains, isotopically non-stationary metabolic flux analysis (INST-MFA) was used to estimate the carbon flux distribution of tktA, 70glpX, and a kanamycin-resistant control (Km), under atmospheric conditions. In addition, untargeted label-free proteomics, which can detect changes in relative enzymatic abundance, was employed to study the possible effects caused by overexpressing each enzyme. Fluxomic and proteomic results indicated a decrease in oxidative pentose phosphate pathway activity when either FBP/SBPase or TK were overexpressed, resulting in increased carbon fixation efficiency. These results are an example of the integration of multiple omic-level experimental techniques and can be used to guide future metabolic engineering efforts to improve performances and efficiencies.

Cucumber mosaic virus-induced gene silencing in banana

Banana (Musa spp.) is one of the world’s most important staple and cash crops. Despite accumulating genetic and transcriptomic data, low transformation efficiency in agronomically important Musa spp. render translational researches in banana difficult by using conventional knockout approaches. To develop tools for translational research in bananas, we developed a virus induced-gene silencing (VIGS) system based on a banana-infecting cucumber mosaic virus (CMV) isolate, CMV 20. CMV 20 genomic RNA 1, 2, and 3, were separately cloned in Agrobacterium pJL89 binary vectors, and a cloning site was introduced on RNA 2 immediately after the 2a open reading frame to insert the gene targeted for silencing. An efficient Agrobacterium inoculation method was developed for banana, which enabled the CMV 20 VIGS vector infection rate to reach 95% in our experiments. CMV 20-based silencing of Musa acuminata cv. Cavendish (AAA group) glutamate 1-semialdehyde aminotransferase (MaGSA) produced a typical chlorotic phenotype and silencing of M. acuminata phytoene desaturase (MaPDS) produced a photobleachnig phenotype. We show this approach efficiently reduced GSA and PDS transcripts to 10% and 18% of the control, respectively. The high infection rate and extended silencing of this VIGS system will provide an invaluable tool to accelerate functional genomic studies in banana.

A Role for PICKLE in the Regulation of Cold and Salt Stress Tolerance in Arabidopsis.

Arabidopsis PICKLE (PKL) is a putative CHD3-type chromatin remodeling factor with important roles in regulating plant growth and development as well as RNA-directed DNA methylation (RdDM). The role of PKL protein in plant abiotic stress response is still poorly understood. Here, we report that PKL is important for cold stress response in Arabidopsis. Loss-of-function mutations in the PKL gene lead to a chlorotic phenotype in seedlings under cold stress, which is caused by the alterations in the transcript levels of some chlorophyll metabolism-related genes. The pkl mutant also exhibits increased electrolyte leakage after freezing treatment. These results suggest that PKL is required for proper chilling and freezing tolerance in plants. Gene expression analysis shows that CBF3, encoding a key transcription factor involved in the regulation of cold-responsive genes, exhibits an altered transcript level in the pkl mutant under cold stress. Transcriptome data also show that PKL regulates the expression of a number of cold-responsive genes, including RD29A, COR15A, and COR15B, possibly through its effect on the expression of CBF3 gene. Mutation in PKL gene also results in decreased cotyledon greening rate and reduced primary root elongation under high salinity. Together, our results suggest that PKL regulates plant responses to cold and salt stress.

Heat treatment alleviates the growth and photosynthetic impairment of transplastomic plants expressing Leishmania infantum Hsp83-Toxoplasma gondii SAG1 fusion protein

Previously, we showed that transplastomic tobacco plants expressing the LiHsp83-SAG1 fusion protein displayed a chlorotic phenotype and growth retardation, while plants expressing the SAG1 and GRA4 antigens alone did not. We conducted a comprehensive examination of the metabolic and photosynthetic parameters that could be affecting the normal growth of LiHsp83-SAG1 plants in order to understand the origin of these pleiotropic effects. These plants presented all photosynthetic pigments and parameters related to PSII efficiency significantly diminished. However, the expression of CHLI, RSSU and LHCa/b genes did not show significant differences between LiHsp83-SAG1 and control plants. Total protein, starch, and soluble sugar contents were also greatly reduced in LiHsp83-SAG1 plants. Since Hsp90 s are constitutively expressed at much higher concentrations at high temperatures, we tested if the fitness of LiHsp83-SAG1 over-expressing LiHsp83 would improve after heat treatment. LiHsp83-SAG1 plants showed an important alleviation of their phenotype and an evident recovery of the PSII function. As far as we know, this is the first report where it is demonstrated that a transplastomic line performs much better at higher temperatures. Finally, we detected that LiHsp83-SAG1 protein could be binding to key photosynthesis-related proteins at 37 °C. Our results suggest that the excess of this molecular chaperone could benefit the plant in a possible heat shock and prevent the expected denaturation of proteins. However, the LiHsp83-SAG1 protein content was weakly decreased in heat-treated plants. Therefore, we cannot rule out that the alleviation observed at 37 °C may be partially due to a reduction of the levels of the recombinant protein.