Jianbin Su

A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes

BackgroundPlant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. In rice, protoplasts are commonly prepared from suspension cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue.ResultsHere, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts.ConclusionsWe show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes.

Lysin Motif–Containing Proteins LYP4 and LYP6 Play Dual Roles in Peptidoglycan and Chitin Perception in Rice Innate Immunity

This work characterizes the roles of two LysM-containing proteins, LYP4 and LYP6, in rice innate immunity. It finds that they act as unique dual-function pattern recognition receptors that can recognize microbe-associated molecular patterns from bacteria and fungi. Plant innate immunity relies on successful detection of microbe-associated molecular patterns (MAMPs) of invading microbes via pattern recognition receptors (PRRs) at the plant cell surface. Here, we report two homologous rice (Oryza sativa) lysin motif–containing proteins, LYP4 and LYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Live cell imaging and microsomal fractionation consistently revealed the plasma membrane localization of these proteins in rice cells. Transcription of these two genes could be induced rapidly upon exposure to bacterial pathogens or diverse MAMPs. Both proteins selectively bound PGN and chitin but not lipopolysaccharide (LPS) in vitro. Accordingly, silencing of either LYP specifically impaired PGN- or chitin- but not LPS-induced defense responses in rice, including reactive oxygen species generation, defense gene activation, and callose deposition, leading to compromised resistance against bacterial pathogen Xanthomonas oryzae and fungal pathogen Magnaporthe oryzae. Interestingly, pretreatment with excess PGN dramatically attenuated the alkalinization response of rice cells to chitin but not to flagellin; vice versa, pretreatment with chitin attenuated the response to PGN, suggesting that PGN and chitin engage overlapping perception components in rice. Collectively, our data support the notion that LYP4 and LYP6 are promiscuous PRRs for PGN and chitin in rice innate immunity.

Evidence for a Role of Chloroplastic m-Type Thioredoxins in the Biogenesis of Photosystem II in Arabidopsis

M-type thioredoxins directly regulate the biogenesis of the photosystem II complex. Chloroplastic m-type thioredoxins (TRX m) are essential redox regulators in the light regulation of photosynthetic metabolism. However, recent genetic studies have revealed novel functions for TRX m in meristem development, chloroplast morphology, cyclic electron flow, and tetrapyrrole synthesis. The focus of this study is on the putative role of TRX m1, TRX m2, and TRX m4 in the biogenesis of the photosynthetic apparatus in Arabidopsis (Arabidopsis thaliana). To that end, we investigated the impact of single, double, and triple TRX m deficiency on chloroplast development and the accumulation of thylakoid protein complexes. Intriguingly, only inactivation of three TRX m genes led to pale-green leaves and specifically reduced stability of the photosystem II (PSII) complex, implying functional redundancy between three TRX m isoforms. In addition, plants silenced for three TRX m genes displayed elevated levels of reactive oxygen species, which in turn interrupted the transcription of photosynthesis-related nuclear genes but not the expression of chloroplast-encoded PSII core proteins. To dissect the function of TRX m in PSII biogenesis, we showed that TRX m1, TRX m2, and TRX m4 interact physically with minor PSII assembly intermediates as well as with PSII core subunits D1, D2, and CP47. Furthermore, silencing three TRX m genes disrupted the redox status of intermolecular disulfide bonds in PSII core proteins, most notably resulting in elevated accumulation of oxidized CP47 oligomers. Taken together, our results suggest an important role for TRX m1, TRX m2, and TRX m4 proteins in the biogenesis of PSII, and they appear to assist the assembly of CP47 into PSII.

Rice MAPK phosphatase IBR5 negatively regulates drought stress tolerance in transgenic Nicotiana tabacum

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are important negative regulators in the MAPK signaling pathways, which play crucial roles in plant growth, regulation of development and response to environment stresses. Several MAPKs have been reported to be involved in the drought stress response, however, there is no evidence for the specific function of MKPs in drought stress. Here, a putative MKP in rice (Oryza sativa), OsIBR5, was characterized. Expression of OsIBR5 was induced by PEG6000, abscisic acid (ABA) and hydrogen peroxide (H(2)O(2)). Overexpression of OsIBR5 in tobacco plants resulted in hypersensitivity to drought and H(2)O(2) treatments. Drought and ABA-induced stomatal closure was significantly reduced in OsIBR5-overexpressing tobacco plants compared with controls. Moreover, OsIBR5 was found to interact with tobacco MAPKs SIPK and WIPK, and drought-induced WIPK activity was impaired in OsIBR5-overexpressing tobacco plants. These results indicated that OsIBR5 is a MKP which was induced by abiotic stresses and decreased tolerance to drought stress in transgenic tobacco plants.

Ferredoxin:Thioredoxin Reductase Is Required for Proper Chloroplast Development and Is Involved in the Regulation of Plastid Gene Expression in Arabidopsis thaliana

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OsPFA-DSP1, a rice protein tyrosine phosphatase, negatively regulates drought stress responses in transgenic tobacco and rice plants

Dephosphorylation plays a pivotal role in regulating plant growth, development and abiotic/biotic stress responses. Here, we characterized a plant and fungi atypical dual-specificity phosphatase (PFA-DSP) subfamily member, OsPFA-DSP1, from rice. OsPFA-DSP1 was determined to be a functional protein tyrosine phosphatase (PTP) in vitro using phosphatase activity assays. Quantitative real-time PCR and GENEVESTIGATOR analysis showed that OsPFA-DSP1 mRNA was induced by drought stress. Transfection of rice protoplasts showed that OsPFA-DSP1 accumulated in both the cytoplasm and nucleus. Ectopic overexpression of OsPFA-DSP1 in tobacco increased sensitivity to drought stress and insensitivity to ABA-induced stomatal closure and inhibition of stomatal opening. Furthermore, overexpression of OsPFA-DSP1 in rice also increased sensitivity to drought stress. These results indicated that OsPFA-DSP1 is a functional PTP and may act as a negative regulator in drought stress responses.

Cu2+ triggers reversible aggregation of a disordered His-rich dehydrin MpDhn12 from Musa paradisiaca.

Copper is an essential nutrient, but it is toxic in excess. Here, we cloned and characterized a His-rich low molecular weight dehydrin from Musa paradisiaca, MpDhn12. Analysis by circular dichroism (CD) spectra and a thermal stability assay showed that MpDhn12 is an intrinsically disordered protein, and immobilized-metal affinity chromatography (IMAC) analysis revealed that MpDhn12 can bind Cu(2+) both in vitro and in vivo. Interestingly, MpDhn12 aggregated under excess Cu(2+) conditions, and the aggregation was reversible and impaired by histidine modification with diethylpyrocarbonate (DEPC), while the disordered structure of another dehydrin ERD14 (as a control) was not changed. Furthermore, MpDhn12 could complement the copper-sensitive phenotype of yeast mutant Δsod1. These results together suggested that MpDhn12 may take part in buffering copper levels through chelation and formation of aggregates in excess Cu(2+) conditions. To the best of our knowledge, it is the first report that a dehydrin interchanged between disordered and aggregated state triggered by copper.

A deficiency in chloroplastic ferredoxin 2 facilitates effective photosynthetic capacity during long-term high light acclimation in Arabidopsis thaliana

Photosynthetic electron transport is the major energy source for cellular metabolism in plants, and also has the potential to generate excess reactive oxygen species that cause irreversible damage to photosynthetic apparatus under adverse conditions. Ferredoxins (Fds), as the electron-distributing hub in the chloroplast, contribute to redox regulation and antioxidant defense. However, the steady-state levels of photosynthetic Fd decrease in plants when they are exposed to environmental stress conditions. To understand the effect of Fd down-regulation on plant growth, we characterized Arabidopsis thaliana plants lacking Fd2 (Fd2-KO) under long-term high light (HL) conditions. Unexpectedly, Fd2-KO plants exhibited efficient photosynthetic capacity and stable thylakoid protein complexes. At the transcriptional level, photoprotection-related genes were up-regulated more in the mutant plants, suggesting that knockout Fd2 lines possess a relatively effective photo-acclimatory responses involving enhanced plastid redox signaling. In contrast to the physiological characterization of Fd2-KO under short-term HL, the plastoquinone pool returned to a relatively balanced redox state via elevated PGR5-dependent cyclic electron flow during extended HL. fd2 pgr5 double mutant plants displayed severely impaired photosynthetic capacity under HL treatment, further supporting a role for PGR5 in adaptation to HL in the Fd2-KO plants. These results suggest potential benefits of reducing Fd levels in plants grown under long-term HL conditions.

Two Homologous Putative Protein Tyrosine Phosphatases, OsPFA-DSP2 and AtPFA-DSP4, Negatively Regulate the Pathogen Response in Transgenic Plants

Protein phosphatases, together with protein kinases, regulate protein phosphorylation and dephosphorylation, and play critical roles in plant growth and biotic stress responses. However, little is known about the biological functions of plant protein tyrosine dual-specificity phosphatase (PFA-DSP) in biotic stresses. Here, we found that OsPFA-DSP2 was mainly expressed in calli, seedlings, roots, and young panicles, and localized in cytoplasm and nucleus. Ectopic overexpression of OsPFA-DSP2 in rice increased sensitivity to Magnaporthe grisea (M. grisea Z1 strain), inhibited the accumulation of hydrogen peroxide (H2O2) and suppressed the expression of pathogenesis-related (PR) genes after fungal infection. Interestingly, transgenic Arabidopsis plants overexpressing AtPFA-DSP4, which is homologous to OsPFA-DSP2, also exhibited sensitivity to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), reduced accumulation of H2O2 and decreased photosynthesic capacity after infection compared with Col-0. These results indicate that OsPFA-DSP2 and AtPFA-DSP4 act as negative regulators of the pathogen response in transgenic plants.

The Chloroplast Min System Functions Differentially in Two Specific Nongreen Plastids in Arabidopsis thaliana

The nongreen plastids, such as etioplasts, chromoplasts, etc., as well as chloroplasts, are all derived from proplastids in the meristem. To date, the Min system members in plants have been identified as regulators of FtsZ-ring placement, which are essential for the symmetrical division of chloroplasts. However, the regulation of FtsZ-ring placement in nongreen plastids is poorly understood. In this study, we investigated the division site placement of nongreen plastids by examining the etioplasts as representative in Arabidopsis Min system mutants. Surprisingly, the shape and number of etioplasts in cotyledons of arc3, arc11 and mcd1 mutants were similar to that observed in wild-type plants, whereas arc12 and parc6 mutants exhibited enlarged etioplasts that were reduced in number. In order to examine nongreen plastids in true leaves, we silenced the ALB3 gene in these Min system mutant backgrounds to produce immature chloroplasts without the thylakoidal network using virus induced gene silencing (VIGS). Interestingly, consistent with our observations in etioplasts, enlarged and fewer nongreen plastids were only detected in leaves of parc6 (VIGS-ALB3) and arc12 (VIGS-ALB3) plants. Further, the FtsZ-ring assembled properly at the midpoint in nongreen plastids of arc3, arc11 and mcd1 (VIGS-ALB3) plants, but organized into multiple rings in parc6 (VIGS-ALB3) and presented fragmented filaments in arc12 (VIGS-ALB3) plants, suggesting that division site placement in nongreen plastids requires fewer components of the plant Min system. Taken together, these results suggest that division site placement in nongreen plastids is different from that in chloroplasts.