scholarly journals The chaperonin 60 protein SlCpn60α1 modulates photosynthesis and photorespiration in tomato

2020 ◽  
Vol 71 (22) ◽  
pp. 7224-7240
Author(s):  
Jie Ye ◽  
Weifang Chen ◽  
Longwei Feng ◽  
Genzhong Liu ◽  
Ying Wang ◽  
...  
Keyword(s):  
Calvin Cycle ◽  
Photosynthetic Electron Transport ◽  
Single Copy ◽  
Chlorophyll Synthesis ◽  
Insertion Mutant ◽  
Loss Of Function ◽  
Virus Induced Gene Silencing ◽  
Dna Insertion ◽  
Dimensional Electrophoresis ◽  
Two Hybrid

Abstract Photosynthesis, an indispensable biological process of plants, produces organic substances for plant growth, during which photorespiration occurs to oxidize carbohydrates to achieve homeostasis. Although the molecular mechanism underlying photosynthesis and photorespiration has been widely explored, the crosstalk between the two processes remains largely unknown. In this study, we isolated and characterized a T-DNA insertion mutant of tomato (Solanum lycopersicum) named yellow leaf (yl) with yellowish leaves, retarded growth, and chloroplast collapse that hampered both photosynthesis and photorespiration. Genetic and expression analyses demonstrated that the phenotype of yl was caused by a loss-of-function mutation resulting from a single-copy T-DNA insertion in chaperonin 60α1 (SlCPN60α1). SlCPN60α1 showed high expression levels in leaves and was located in both chloroplasts and mitochondria. Silencing of SlCPN60α1using virus-induced gene silencing and RNA interference mimicked the phenotype of yl. Results of two-dimensional electrophoresis and yeast two-hybrid assays suggest that SlCPN60α1 potentially interacts with proteins that are involved in chlorophyll synthesis, photosynthetic electron transport, and the Calvin cycle, and further affect photosynthesis. Moreover, SlCPN60α1 directly interacted with serine hydroxymethyltransferase (SlSHMT1) in mitochondria, thereby regulating photorespiration in tomato. This study outlines the importance of SlCPN60α1 for both photosynthesis and photorespiration, and provides molecular insights towards plant genetic improvement.

scholarly journals Expression of α-DIOXYGENASE 1 in Tomato and Arabidopsis Contributes to Plant Defenses Against Aphids

2013 ◽  
Vol 26 (8) ◽  
pp. 977-986 ◽  
Author(s):  
Carlos Augusto Avila ◽  
Lirio Milenka Arevalo-Soliz ◽  
Argelia Lorence ◽  
Fiona L. Goggin
Keyword(s):  
Green Peach Aphid ◽  
Macrosiphum Euphorbiae ◽  
Insertion Mutant ◽  
Plant Signaling ◽  
Virus Induced Gene Silencing ◽  
Basal Resistance ◽  
Potato Aphid ◽  
Dna Insertion ◽  
Mutant Lines ◽  
Increased Susceptibility

Plant α-dioxygenases (α-DOX) are fatty acid–hydroperoxidases that contribute to the synthesis of oxylipins, a diverse group of compounds primarily generated through oxidation of linoleic (LA) and linolenic acid (LNA). Oxylipins are implicated in plant signaling against biotic and abiotic stresses. We report here that the potato aphid (Macrosiphum euphorbiae) induces Slα-DOX1 but not Slα-DOX2 expression in tomato (Solanum lycopersicum). Slα-DOX1 upregulation by aphids does not require either jasmonic acid (JA) or salicylic acid (SA) accumulation, since tomato mutants deficient in JA (spr2, acx1) or SA accumulation (NahG) still show Slα-DOX1 induction. Virus-induced gene silencing of Slα-DOX1 enhanced aphid population growth in wild-type (WT) plants, revealing that Slα-DOX1 contributes to basal resistance to aphids. Moreover, an even higher percent increase in aphid numbers occurred when Slα-DOX1 was silenced in spr2, a mutant line characterized by elevated LA levels, decreased LNA, and enhanced aphid resistance as compared with WT. These results suggest that aphid reproduction is influenced by oxylipins synthesized from LA by Slα-DOX1. In agreement with our experiments in tomato, two independent α-dox1 T-DNA insertion mutant lines in Arabidopsis thaliana also showed increased susceptibility to the green peach aphid (Myzus persicae), indicating that the role α-DOX is conserved in other plant-aphid interactions.

scholarly journals Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus

2021 ◽  
Vol 7 (4) ◽  
pp. 252
Author(s):  
Rundong Liu ◽  
Wonyong Kim ◽  
Jaycee Augusto Paguirigan ◽  
Min-Hye Jeong ◽  
Jae-Seoun Hur
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Incubation Temperature ◽  
Single Copy ◽  
Insertion Mutant ◽  
Symbiotic Association ◽  
Slow Growth Rate ◽  
Cultivation Period ◽  
C Terminus ◽  
Genetic Mechanisms ◽  
Dna Insertion

Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from Cladonia macilenta. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with Agrobacterium cells, and incubation temperature, were tested to identify an optimized ATMT condition for the C. macilenta mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in C. macilenta, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis.

scholarly journals mRNA Surveillance Complex PELOTA-HBS1 Regulates Phosphoinositide-Dependent Protein Kinase1 and Plant Growth

2021 ◽  
Author(s):  
Wei Kong ◽  
Shutang Tan ◽  
Qing Zhao ◽  
De-Li Lin ◽  
Zhi-Hong Xu ◽  
...  
Keyword(s):  
Quality Control ◽  
Messenger Rna ◽  
Loss Of Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Developmental Defects ◽  
Mrna Surveillance ◽  
Dna Insertion ◽  
Dependent Protein ◽  
Heterodimeric Complex ◽  
Severe Growth

Abstract The quality control system for messenger RNA (mRNA) is fundamental for cellular activities in eukaryotes. To elucidate the molecular mechanism of 3’-Phosphoinositide-Dependent Protein Kinase1 (PDK1), a master regulator that is essential throughout eukaryotic growth and development, we employed a forward genetic approach to screen for suppressors of the loss-of-function T-DNA insertion double mutant pdk1.1 pdk1.2 in Arabidopsis thaliana. Notably, the severe growth attenuation of pdk1.1 pdk1.2 was rescued by sop21 (suppressor of pdk1.1 pdk1.2), which harbours a loss-of-function mutation in PELOTA1 (PEL1). PEL1 is a homologue of mammalian PELOTA and yeast (Saccharomyces cerevisiae) DOM34p, which each form a heterodimeric complex with the GTPase HBS1 (HSP70 SUBFAMILY B SUPPRESSOR1, also called SUPERKILLER PROTEIN7, SKI7), a protein that is responsible for ribosomal rescue and thereby assures the quality and fidelity of mRNA molecules during translation. Genetic analysis further revealed that a dysfunctional PEL1-HBS1 complex failed to degrade the T-DNA-disrupted PDK1 transcripts, which were truncated but functional, and thus rescued the growth and developmental defects of pdk1.1 pdk1.2. Our studies demonstrated the functionality of a homologous PELOTA-HBS1 complex and identified its essential regulatory role in plants, providing insights into the mechanism of mRNA quality control.

scholarly journals A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana

2021 ◽  
Vol 118 (17) ◽  
pp. e2021293118
Author(s):  
Yonglun Zeng ◽  
Baiying Li ◽  
Changyang Ji ◽  
Lei Feng ◽  
Fangfang Niu ◽  
...  
Keyword(s):  
Stress Responses ◽  
Higher Plants ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Secretory Proteins ◽  
Insertion Mutant ◽  
Intermediate Metabolites ◽  
Dna Insertion ◽  
Autophagy Pathway ◽  
Copii Vesicles

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

scholarly journals Plastid-Localized EMB2726 Is Involved in Chloroplast Biogenesis and Early Embryo Development in Arabidopsis

2021 ◽  
Vol 12 ◽  
Author(s):  
Chuanling Li ◽  
Jian-Xiu Shang ◽  
Chenlei Qiu ◽  
Baowen Zhang ◽  
Jinxue Wang ◽  
...  
Keyword(s):  
Chloroplast Development ◽  
Developmental Process ◽  
Higher Plants ◽  
Critical Role ◽  
Elongation Factor ◽  
Early Embryo ◽  
The Body ◽  
Insertion Mutant ◽  
Cell Stage ◽  
Dna Insertion

Embryogenesis is a critical developmental process that establishes the body organization of higher plants. During this process, the biogenesis of chloroplasts from proplastids is essential. A failure in chloroplast development during embryogenesis can cause morphologically abnormal embryos or embryonic lethality. In this study, we isolated a T-DNA insertion mutant of the Arabidopsis gene EMBRYO DEFECTIVE 2726 (EMB2726). Heterozygous emb2726 seedlings produced about 25% albino seeds with embryos that displayed defects at the 32-cell stage and that arrested development at the late globular stage. EMB2726 protein was localized in chloroplasts and was expressed at all stages of development, such as embryogenesis. Moreover, the two translation elongation factor Ts domains within the protein were critical for its function. Transmission electron microscopy revealed that the cells in emb2726 embryos contained undifferentiated proplastids and that the expression of plastid genome-encoded photosynthesis-related genes was dramatically reduced. Expression studies of DR5:GFP, pDRN:DRN-GFP, and pPIN1:PIN1-GFP reporter lines indicated normal auxin biosynthesis but altered polar auxin transport. The expression of pSHR:SHR-GFP and pSCR:SCR-GFP confirmed that procambium and ground tissue precursors were lacking in emb2726 embryos. The results suggest that EMB2726 plays a critical role during Arabidopsis embryogenesis by affecting chloroplast development, possibly by affecting the translation process in plastids.

scholarly journals FIGNL1 associates with KIF1Bβ and BICD1 to restrict dynein transport velocity during axon navigation

2019 ◽  
Vol 218 (10) ◽  
pp. 3290-3306 ◽  
Author(s):  
Melody Atkins ◽  
Laïla Gasmi ◽  
Valérie Bercier ◽  
Céline Revenu ◽  
Filippo Del Bene ◽  
...  
Keyword(s):  
Molecular Motor ◽  
Motor Axon ◽  
Regulatory Mechanisms ◽  
Axon Pathfinding ◽  
Loss Of Function ◽  
Yeast Two Hybrid ◽  
Neuronal Connectivity ◽  
Transport Velocity ◽  
Bidirectional Transport ◽  
Two Hybrid

Neuronal connectivity relies on molecular motor-based axonal transport of diverse cargoes. Yet the precise players and regulatory mechanisms orchestrating such trafficking events remain largely unknown. We here report the ATPase Fignl1 as a novel regulator of bidirectional transport during axon navigation. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we showed that Fignl1 binds the kinesin Kif1bβ and the dynein/dynactin adaptor Bicaudal D-1 (Bicd1) in a molecular complex including the dynactin subunit dynactin 1. Fignl1 colocalized with Kif1bβ and showed bidirectional mobility in zebrafish axons. Notably, Kif1bβ and Fignl1 loss of function similarly altered zebrafish motor axon pathfinding and increased dynein-based transport velocity of Rab3 vesicles in these navigating axons, pinpointing Fignl1/Kif1bβ as a dynein speed limiter complex. Accordingly, disrupting dynein/dynactin activity or Bicd1/Fignl1 interaction induced motor axon pathfinding defects characteristic of Fignl1 gain or loss of function, respectively. Finally, pharmacological inhibition of dynein activity partially rescued the axon pathfinding defects of Fignl1-depleted larvae. Together, our results identify Fignl1 as a key dynein regulator required for motor circuit wiring.

scholarly journals Comparative Proteomic Analysis Reveals Elevated Capacity for Photosynthesis in Polyphenol Oxidase Expression-Silenced Clematis terniflora DC. Leaves

2018 ◽  
Vol 19 (12) ◽  
pp. 3897 ◽  
Author(s):  
Xi Chen ◽  
Bingxian Yang ◽  
Wei Huang ◽  
Tantan Wang ◽  
Yaohan Li ◽  
...  
Keyword(s):  
Polyphenol Oxidase ◽  
Agricultural Production ◽  
Molecular Mechanisms ◽  
Calvin Cycle ◽  
Virus Induced Gene Silencing ◽  
Light Reaction ◽  
Plant Photosynthesis ◽  
Mapman Analysis ◽  
Glycolysis Process

Polyphenol oxidase (PPO) catalyzes the o-hydroxylation of monophenols and oxidation of o-diphenols to quinones. Although the effects of PPO on plant physiology were recently proposed, little has been done to explore the inherent molecular mechanisms. To explore the in vivo physiological functions of PPO, a model with decreased PPO expression and enzymatic activity was constructed on Clematis terniflora DC. using virus-induced gene silencing (VIGS) technology. Proteomics was performed to identify the differentially expressed proteins (DEPs) in the model (VC) and empty vector-carrying plants (VV) untreated or exposed to high levels of UV-B and dark (HUV-B+D). Following integration, it was concluded that the DEPs mainly functioned in photosynthesis, glycolysis, and redox in the PPO silence plants. Mapman analysis showed that the DEPs were mainly involved in light reaction and Calvin cycle in photosynthesis. Further analysis illustrated that the expression level of adenosine triphosphate (ATP) synthase, the content of chlorophyll, and the photosynthesis rate were increased in VC plants compared to VV plants pre- and post HUV-B+D. These results indicate that the silence of PPO elevated the plant photosynthesis by activating the glycolysis process, regulating Calvin cycle and providing ATP for energy metabolism. This study provides a prospective approach for increasing crop yield in agricultural production.

scholarly journals MAD3 Encodes a Novel Component of the Spindle Checkpoint Which Interacts with Bub3p, Cdc20p, and Mad2p

2000 ◽  
Vol 148 (5) ◽  
pp. 871-882 ◽  
Author(s):  
Kevin G. Hardwick ◽  
Raymond C. Johnston ◽  
Dana L. Smith ◽  
Andrew W. Murray
Keyword(s):  
Cell Cycle ◽  
Sequence Analysis ◽  
Protein Kinase ◽  
Nuclear Protein ◽  
Budding Yeast ◽  
Spindle Checkpoint ◽  
Terminal Region ◽  
Loss Of Function ◽  
Checkpoint Proteins ◽  
Two Hybrid

We show that MAD3 encodes a novel 58-kD nuclear protein which is not essential for viability, but is an integral component of the spindle checkpoint in budding yeast. Sequence analysis reveals two regions of Mad3p that are 46 and 47% identical to sequences in the NH2-terminal region of the budding yeast Bub1 protein kinase. Bub1p is known to bind Bub3p (Roberts et al. 1994) and we use two-hybrid assays and coimmunoprecipitation experiments to show that Mad3p can also bind to Bub3p. In addition, we find that Mad3p interacts with Mad2p and the cell cycle regulator Cdc20p. We show that the two regions of homology between Mad3p and Bub1p are crucial for these interactions and identify loss of function mutations within each domain of Mad3p. We discuss roles for Mad3p and its interactions with other spindle checkpoint proteins and with Cdc20p, the target of the checkpoint.

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