Wann-Neng Jane

LZF1, a HY5-regulated transcriptional factor, functions in Arabidopsis de-etiolation

We surveyed differential gene expression patterns during early photomorphogenesis in both wild-type and mutant Arabidopsis defective in HY5, an influential positive regulator of the responses of gene expression to a light stimulus, to identify light-responsive genes whose expression was HY5 dependent. These gene-expression data identified light-regulated zinc finger protein 1 (LZF1), a gene encoding a previously uncharacterized C2C2-CO B-box transcriptional regulator. HY5 has positive trans-activating activity toward LZF1 and binding affinity to LZF1 promoter in vivo. HY5 is needed but not sufficient for the induction of LZF1 expression. Anthocyanin content is significantly diminished in lzf1 under far red, which is the most efficient light for the induction of LZF1. The expression of PAP1/MYB75 is elevated in plants overexpressing LZF1, which leads to the hyperaccumulation of anthocyanin in transgenic Arabidopsis. The transition from etioplast to chloroplast and the accumulation of chlorophyll were notably compromised in the lzf1 mutant. We provide molecular evidence that LZF1 influences chloroplast biogenesis and function via regulating genes encoding chloroplast proteins. In the absence of HY5, mutation of LZF1 leads to further reduced light sensitivity for light-regulated inhibition of hypocotyl elongation and anthocyanin and chlorophyll accumulation. Our data indicate that LZF1 is a positive regulator functioning in Arabidopsis de-etiolation.

Arabidopsis thaliana CENTRORADIALIS homologue (ATC) acts systemically to inhibit floral initiation in Arabidopsis

Floral initiation is orchestrated by systemic floral activators and inhibitors. This remote-control system may integrate environmental cues to modulate floral initiation. Recently, FLOWERING LOCUS T (FT) was found to be a florigen. However, the identity of systemic floral inhibitor or anti-florigen remains to be elucidated. Here we show that Arabidopsis thaliana CENTRORADIALIS homologue (ATC), an Arabidopsis FT homologue, may act in a non-cell autonomous manner to inhibit floral initiation. Analysis of the ATC null mutant revealed that ATC is a short-day-induced floral inhibitor. Cell type-specific expression showed that companion cells and apex that express ATC are sufficient to inhibit floral initiation. Histochemical analysis showed that the promoter activity of ATC was mainly found in vasculature but under the detection limit in apex, a finding that suggests that ATC may move from the vasculature to the apex to influence flowering. Consistent with this notion, Arabidopsis seedling grafting experiments demonstrated that ATC moved over a long distance and that floral inhibition by ATC is graft transmissible. ATC probably antagonizes FT activity, because both ATC and FT interact with FD and affect the same downstream meristem identity genes APETALA1, in an opposite manner. Thus, photoperiodic variations may trigger functionally opposite FT homologues to systemically influence floral initiation.

Role of the Putative Osmosensor Arabidopsis Histidine Kinase1 in Dehydration Avoidance and Low-Water-Potential Response

Summary: AHK1 mutants had increased stomatal density and faster leaf water loss; however, AHK1 mutants were not more sensitive to controlled low water potential stress and not impaired in abscisic acid, Pro, or osmoregulatory solute accumulation, thus indicating that AHK1 is not the main plant osmosensor controlling these drought tolerance-associated phenotypes. The molecular basis of plant osmosensing remains unknown. Arabidopsis (Arabidopsis thaliana) Histidine Kinase1 (AHK1) can complement the osmosensitivity of yeast (Saccharomyces cerevisiae) osmosensor mutants lacking Synthetic Lethal of N-end rule1 and SH3-containing Osmosensor and has been proposed to act as a plant osmosensor. We found that ahk1 mutants in either the Arabidopsis Nossen-0 or Columbia-0 background had increased stomatal density and stomatal index consistent with greater transpirational water loss. However, the growth of ahk1 mutants was not more sensitive to controlled moderate low water potential (ψw) or to salt stress. Also, ahk1 mutants had increased, rather than reduced, solute accumulation across a range of low ψw severities. ahk1 mutants had reduced low ψw induction of Δ1-Pyrroline-5-Carboxylate Synthetase1 (P5CS1) and 9-cis-Epoxycarotenoid Dioxygenase3, which encode rate-limiting enzymes in proline and abscisic acid (ABA) synthesis, respectively. However, neither Pro nor ABA accumulation was reduced in ahk1 mutants at low ψw. P5CS1 protein level was not reduced in ahk1 mutants. This indicated that proline accumulation was regulated in part by posttranscriptional control of P5CS1 that was not affected by AHK1. Expression of AHK1 itself was reduced by low ψw, in contrast to previous reports. These results define a role of AHK1 in controlling stomatal density and the transcription of stress-responsive genes. These phenotypes may be mediated in part by reduced ABA sensitivity. More rapid transpiration and water depletion can also explain the previously reported sensitivity of ahk1 to uncontrolled soil drying. The unimpaired growth, ABA, proline, and solute accumulation of ahk1 mutants at low ψw suggest that AHK1 may not be the main plant osmosensor required for low ψw tolerance.

Transplastomic Nicotiana benthamiana plants expressing multiple defence genes encoding protease inhibitors and chitinase display broad‐spectrum resistance against insects, pathogens and abiotic stresses

Plastid engineering provides several advantages for the next generation of transgenic technology, including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. With the goal of generating transplastomic plants with multiresistance against both phytopathogens and insects, a construct containing a monocistronic patterned gene stack was transformed into Nicotiana benthamiana plastids harbouring sweet potato sporamin, taro cystatin and chitinase from Paecilomyces javanicus. Transplastomic lines were screened and characterized by Southern/Northern/Western blot analysis for the confirmation of transgene integration and respective expression level. Immunogold localization analyses confirmed the high level of accumulation proteins that were specifically expressed in leaf and root plastids. Subsequent functional bioassays confirmed that the gene stacks conferred a high level of resistance against both insects and phytopathogens. Specifically, larva of Spodoptera litura and Spodoptera exigua either died or exhibited growth retardation after ingesting transplastomic plant leaves. In addition, the inhibitory effects on both leaf spot diseases caused by Alternaria alternata and soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum were markedly observed. Moreover, tolerance to abiotic stresses such as salt/osmotic stress was highly enhanced. The results confirmed that the simultaneous expression of sporamin, cystatin and chitinase conferred a broad spectrum of resistance. Conversely, the expression of single transgenes was not capable of conferring such resistance. To the best of our knowledge, this is the first study to demonstrate an efficacious stacked combination of plastid-expressed defence genes which resulted in an engineered tolerance to various abiotic and biotic stresses.

Morphological changes of Vibrio parahaemolyticus under cold and starvation stresses.

This study addresses morphological changes of Vibrio parahaemolyticus cells during the initial period of stress. Drastic changes occurred in cells under starvation with/without concomitant cold treatment for one to a few days. Under carbon starvation, rod-shaped cells became coccoid by reductive division, which was influenced by glucose/amino acid in the culture medium. Under concomitant cold and carbon starvation, cells entered the viable but nonculturable state and their shape changed from rod-like to coccoid with waning-shape and various aberrantly intermediates, such as club-shaped, budding, club-shaped with budding, dumbbell-shaped and rabbit-ear-like-shaped. Electron microscopy revealed characteristic features in the cells that were concomitantly stressed with cold and starved, such as densely stained peripheral part and lightly stained central part of cytoplasm, and a thick peptidoglycan cell wall in the temperature-upshifted cells. The expression of cytoskeleton genes, mreB, minE and ftsZ, was determined in a real-time quantitative reverse transcription-polymerase chain reaction, and results revealed a substantial decline in expression in cells under starvation (to 0.25 to 0.64 times that of the control) in the first 2 to 3 h, and a rapid upregulated expression in recovering cells. Alteration of the morphology of cells under such stresses may be associated with the expression of these cytoskeleton genes. This work demonstrates the uniqueness of concomitant cold and starvation treatment in V. parahaemolyticus. The details of the morphological changes may facilitate understanding of stress adaptation in bacteria.

Structural and functional characterizations of mung bean mitochondrial nucleoids

Mitochondrial nucleoids isolated from mung bean seedlings exhibited a chromatin-like structure associated with a membrane component. A similar structure, which underwent discrete changes during cotyledon development, was identified in situ. Isolated nucleoids consisted of essentially the same phospholipids, including cardiolipin, as whole mitochondria and proteins of inner- and outer-mitochondrial-membrane origin. Actin was consistently found with mitochondrial nucleoids prepared with different detergent concentrations. Formaldehyde cross-linking of cytochalasin B- and proteinase K-treated mitochondria further revealed that actin was associated with DNA in nucleoids. Mitochondrial nucleoids were self-sufficient in directing DNA synthesis in vitro in a pattern mimicking mtDNA synthesis in isolated mitochondria. In pulse-field gel electrophoresis, newly synthesized mtDNA separated into two major components, well-bound and fast-moving forms. Nucleoids DNA synthesis was resistant to aphidicolin but sensitive to N-ethylmaleimide, which indicates that a γ-type DNA polymerase was responsible for this activity. Mitochondrial nucleoids were capable of self-directed RNA transcription in a non-random fashion in vitro. Consistent with and complementary to results from fungi and human cells done mostly in situ, our present work helps to establish the important paradigm that mitochondrial nucleoids in eukaryotes are more than mere mtDNA compaction and segregation entities but are centers of mtDNA maintenance and expression.

Peptide-mediated liposomal Doxorubicin enhances drug delivery efficiency and therapeutic efficacy in animal models.

Lung cancer ranks among the most common malignancies, and is the leading cause of cancer-related mortality worldwide. Chemotherapy for lung cancer can be made more specific to tumor cells, and less toxic to normal tissues, through the use of ligand-mediated drug delivery systems. In this study, we investigated the targeting mechanism of the ligand-mediated drug delivery system using a peptide, SP5-2, which specifically binds to non-small cell lung cancer (NSCLC) cells. Conjugation of SP5-2 to liposomes enhanced the amount of drug delivered directly into NSCLC cells, through receptor-mediated endocytosis. Functional SP5-2 improved the therapeutic index of Lipo-Dox by enhancing therapeutic efficacy, reducing side effects, and increasing the survival rate of tumor-bearing mice in syngenic, metastatic and orthotopic animal models. Accumulation of SP5-2-conjugated liposomal doxorubicin (SP5-2-LD) in tumor tissues was 11.2-fold higher than that of free doxorubicin, and the area under the concentration-time curve (AUC0–72 hours) was increased 159.2-fold. Furthermore, the experiment of bioavailability was assessed to confirm that SP5-2 elevates the uptake of the liposomal drugs by the tumor cells in vivo. In conclusion, the use of SP5-2-conjugated liposomes enhances pharmacokinetic properties, improves efficacy and safety profiles, and allows for controlled biodistribution and drug release.

Isolation and characterization of an isocitrate lyase gene from senescent leaves of sweet potato (Ipomoea batatas cv. Tainong 57).

Summary Isocitrate lyase and malate synthase are two key enzymes of the glyoxylate cycle, and are associated with lipid degradation and gluconeogenesis in microorganisms, plants, nematodes, and animals. Although genes of isocitrate lyase have been cloned from seedling cotyledons of several plants, none were isolated from the senescent leaves. In this study, sweet potato yellow leaves with disassembled chloroplast thylakoids and remarkable osmiophilic globule accumulation in stroma were used as material. Using both PCR-based subtractive hybridization and RACE PCR, a 2.1 kb full-length sp/CL cDNA was cloned. The open reading frame contained 1728 nucleotides (576 amino acids) and exhibited more than 75% sequence identity with plant cotyledon isocitrate lyases of tomato, upland cotton, cucumber, castor bean, rape, soybean, and loblolly pine. The sp/CL-encoded protein contained the Leu-169, Lys-170, Pro-171 (LKP) and Thr-210, Lys-211, Lys-212 (TKK) motifs, with a reported substrate binding domain function, as well as a putative peroxisomal targeting signal (PTS) Ala-Arg-Met (ARM) tripeptide at the C-terminus. Its mANA accumulated exclusively in the senescing and completely yellow leaves, but not in the green leaves, roots, or stems. Hence, the sweet potato sp/CL is the first isocitrate lyase isolated from senescent leaves. The data may provide molecular evidence to support the notion that glyoxylate cycle, a metabolic pathway utilized in cotyledons for postgerminative growth of oilseeds, is also involved in lipid degradation and gluconeogenesis of senescent leaves.

Chloroplast genome aberration in micropropagation-derived albino Bambusa edulis mutants, ab1 and ab2

Two albino mutants (ab1 and ab2) have been derived from long-term shoot proliferation of Bambusa edulis. Based on transmission electronic microscopy data, the chloroplasts of these mutants were abnormal. To study the mutation of gene regulation in the aberrant chloroplasts, we designed 19 pairs of chloroplast-encoded gene primers for genomic and RT-PCR. Only putative NAD(P)H-quinone oxidoreductase chain 4L (ndhE; DQ908943) and ribosomal protein S7 (rps7; DQ908931) were conserved in both the mutant and wild-type plants. The deletions in the chloroplast genome of these two mutants were different: nine genes were deleted in the chloroplast genomic aberration in ab1 and 11 genes in ab2. The chloroplast genes, NAD(P)H-quinone oxidoreductase chain 4 (ndhD; DQ908944), chloroplast 50S ribosomal protein L14 (rpl14; DQ908934), and ATP synthase beta chain (atpB; DQ908948) were abnormal in both mutants. The gene expressions of 18 of these 20 genes were correlated with their DNA copy number. The two exceptions were: ATP synthase CF0 A chain (atpI; DQ908946), whose expression in both mutants was not reduced even though the copy number was reduced; ribosomal protein S19 (rps19; DQ908949), whose expression was reduced or it was not expressed at all even though there was no difference in genomic copy number between the wild-type and mutant plants. The genomic PCR results showed that chloroplast genome aberrations do occur in multiple shoot proliferation, and this phenomenon may be involved in the generation of albino mutants.

De novo centriole formation in human cells is error-prone and does not require SAS-6 self-assembly

Vertebrate centrioles normally propagate through duplication, but in the absence of preexisting centrioles, de novo synthesis can occur. Consistently, centriole formation is thought to strictly rely on self-assembly, involving self-oligomerization of the centriolar protein SAS-6. Here, through reconstitution of de novo synthesis in human cells, we surprisingly found that normal looking centrioles capable of duplication and ciliation can arise in the absence of SAS-6 self-oligomerization. Moreover, whereas canonically duplicated centrioles always form correctly, de novo centrioles are prone to structural errors, even in the presence of SAS-6 self-oligomerization. These results indicate that centriole biogenesis does not strictly depend on SAS-6 self-assembly, and may require preexisting centrioles to ensure structural accuracy, fundamentally deviating from the current paradigm. DOI: http://dx.doi.org/10.7554/eLife.10586.001