The Arabidopsis voltage-dependent anion channel 2 is required for plant growth

The voltage-dependent anion channels (VDACs), known as a major group of outer mitochondrial membrane proteins, are present in all eukaryotic species. In mammalian cells, they have been established as a key player in mitochondrial metabolism and apoptosis regulation. By contrast, little is known about the function of plant VDACs. Recently, we performed functional analysis of all VDAC gene members in Arabidopsis thaliana, and revealed that each AtVDAC member has a specialized function. Especially, in spite of similar subcellular localization and expression profiling of AtVDAC2 and AtVDAC4, both the T-DNA insertion knockout mutants of them, vdac2–2 and vdac4–2, showed severe growth retardation. These results suggest that AtVDAC2 and AtVDAC4 proteins clearly have distinct functions. Here, we introduced the AtVDAC2 gene into the vdac2–2 mutant, and demonstrated that the miniature phenotype of vdac2–2 plant is abolished by AtVDAC2 expression.     

Flowering‐mediated root‐fungus symbiosis loss is related to jasmonate‐dependent root soluble sugar deprivation

The role of flowering in root‐fungal symbiosis is not well understood. Because flowering and fungal symbionts are supported by carbohydrates, we hypothesized that flowering modulates root‐beneficial fungal associations through alterations in carbohydrate metabolism and transport. We monitored fungal colonization and soluble sugars in the roots of Arabidopsis thaliana following inoculation with a mutualistic fungus Phomopsis liquidambari across different plant developmental stages. Jasmonate signalling of wild‐type plants, sugar transport, and root invertase of wild‐type and jasmonate‐insensitive plants were exploited to assess whether and how jasmonate‐dependent sugar dynamics are involved in flowering‐mediated fungal colonization alterations. We found that flowering restricts root‐fungal colonization and activates root jasmonate signalling upon fungal inoculation. Jasmonates reduce the constitutive and fungus‐induced accumulation of root glucose and fructose at the flowering stage. Further experiments with sugar transport and metabolism mutant lines revealed that root glucose and fructose positively influence fungal colonization. Diurnal, jasmonate‐dependent inhibitions of sugar transport and soluble invertase activity were identified as likely mechanisms for flowering‐mediated root sugar depletion upon fungal inoculation. Collectively, our results reveal that flowering drives root‐fungus cooperation loss, which is related to jasmonate‐dependent root soluble sugar depletion. Limiting the spread of root‐fungal colonization may direct more resources to flower development.     

THESEUS1 is involved in tunicamycin-induced root growth inhibition,ectopic lignin deposition,and cell wall damage-induced unfolded protein response

Endoplasmic reticulum (ER) stress activates unfolded protein responses (UPRs), such as promoting protein folding under the control of specific gene expression. Our previous study showed that ER stress induced by ER stress inducers such as tunicamycin (Tm), an inhibitor of N-linked glycan synthesis, causes ectopic lignin deposition in Arabidopsis roots, but the relationship between UPR and ectopic lignin deposition remains unclear. The receptor-like kinase THESEUS1 (THE1) has been shown to sense cell wall damage (CWD) induced in Arabidopsis by cellulose synthase inhibitors such as isoxaben (ISO) and to activate ectopic lignin deposition. In this study, we assessed the involvement of THE1 in ectopic lignin deposition caused by the ER stress inducer Tm. The loss-of-function mutation of THE1, the1-3, suppressed Tm-induced root growth inhibition and ectopic lignin deposition, revealing that THE1 is involved in root growth defects and ectopic lignin deposition caused by ER stress. Similarly, ISO treatment induced ectopic lignin deposition as well as the expression of the UPR marker genes binding protein 3 (BiP3) and ER-localized DnaJ 3b (ERdj3b). Conversely, in the the1-3 mutant, ISO-induced ectopic lignin deposition and the expression of BiP3 and ERdj3b were suppressed. These results showed that THE1 is involved in not only root growth inhibition and ectopic lignin deposition caused by ER stress but also CWD-induced UPR.     

Cdc5L interacts with ATR and is required for the S‐phase cell‐cycle checkpoint

Cell division cycle 5‐like protein (Cdc5L) is a core component of the putative E3 ubiquitin ligase complex containing Prp19/Pso4, Plrg1 and Spf27. This complex has been shown to have a role in pre‐messenger RNA splicing from yeast to humans; however, more recent studies have described a function for this complex in the cellular response to DNA damage. Here, we show that Cdc5L interacts physically with the cell‐cycle checkpoint kinase ataxia‐telangiectasia and Rad3‐related (ATR). Depletion of Cdc5L by RNA‐mediated interference methods results in a defective S‐phase cell‐cycle checkpoint and cellular sensitivity in response to replication‐fork blocking agents. Furthermore, we show that Cdc5L is required for the activation of downstream effectors or mediators of ATR checkpoint function such as checkpoint kinase 1 (Chk1), cell cycle checkpoint protein Rad 17 (Rad17) and Fanconi anaemia complementation group D2 protein (FancD2). In addition, we have mapped the ATR‐binding region in Cdc5L and show that a deletion mutant that is unable to interact with ATR is defective in the rescue of the checkpoint deficiency in Cdc5L‐depleted cells. These findings show a new function for Cdc5L in the regulation of the ATR‐mediated cell‐cycle checkpoint in response to genotoxic agents.     

RAB GTPASE HOMOLOG 8D is required for the maintenance of both the root stem cell niche and the meristem

Previous studies have suggested that the plastid translation elongation factor, elongation factor thermo unstable (EF-Tu), encoded by RAB GTPASE HOMOLOG 8D (RAB8D) is essential for plant growth. Here, through analyzing the root phenotypes of two knock-down alleles of RAB8D (rab8d-1 and rab8d-2), we further revealed a vital role for RAB8D in primary root development through the maintenance of both the stem cell niche (SCN) and the meristem. Our results showed that RAB8D deficiency affects the root auxin response and SCN maintenance signaling. RAB8D interacts with GENOMES UNCOUPLED 1 (GUN1) in vivo. Further analysis revealed that GUN1 is over-accumulated and is required for both stem cell death and maintenance of root architecture in rab8d Arabidopsis mutants. The ATAXIA-TELANGIECTASIA-MUTATED (ATM)–SUPPRESSOR OF GAMMA RESPONSE 1 pathway is involved in the regulation of root meristem size through upregulating SIAMESE-RELATED 5 expression in the rab8d-2 allele. Moreover, ETHYLENE RESPONSE FACTOR 115 is highly expressed in rab8d-2, which plays a role in further quiescent center division. Our observations not only characterized the role of RAB8D in root development, but also uncovered functions of GUN1 and ATM in response to plastid EF-Tu deficiency.     

The TUMOROUS SHOOT DEVELOPMENT2 gene of Arabidopsis encoding a putative methyltransferase is required for cell adhesion and co-ordinated plant development

Mutations in the TUMOROUS SHOOT DEVELOPMENT2 (TSD2) gene reduce cell adhesion, and in strongly affected individuals cause non-coordinated shoot development that leads to disorganized tumor-like growth in vitro. tsd2 mutants showed increased activity of axial meristems, reduced root growth and enhanced de-etiolation. The expression domains of the shoot meristem marker genes KNAT1 and KNAT2 were enlarged in the mutant background. Soil-grown tsd2 mutants were dwarfed, but overall showed morphology similar to that of the wild-type (WT). The TSD2 gene was identified by map-based cloning. It encodes a novel 684 amino acid polypeptide containing a single membrane-spanning domain in the N-terminal part and S-adenosyl-l-methionine binding and methyltransferase domains in the C-terminal part. Expression of a TSD2:GUS reporter gene was detected mainly in meristems and young tissues. A green fluorescent protein-tagged TSD2 protein localized to the Golgi apparatus. The cell-adhesion defects indicated altered pectin properties, and we hypothesize that TSD2 acts as a pectin methyltransferase. However, analyses of the cell-wall composition revealed no significant differences of the monosaccharide composition, the uronic acid content and the overall degree of pectin methylesterification between tsd2 and WT. The findings support a function of TSD2 as a methyltransferase, with an essential role in cell adhesion and coordinated plant development.     

BUD2, encoding an S-adenosylmethionine decarboxylase, is required for Arabidopsis growth and development

Polyamines are implicated in regulating various developmental processes in plants, but their exact roles and how they govern these processes still remain elusive. We report here an Arabidopsis bushy and dwarf mutant, bud2, which results from the complete deletion of one member of the small gene family that encodes S-adenosylmethionine decarboxylases （SAMDCs） necessary for the formation of the indispensable intermediate in the polyamine biosynthetic pathway. The bud2 plant has enlarged vascular systems in inflorescences, roots, and petioles, and an altered homeostasis ofpolyamines. The double mutant of bud2 and samdcl, a knockdown mutant of another SAMDC member, is embryo lethal, demonstrating that SAMDCs are essential for plant embryogenesis. Our results suggest that polyamines are required for the normal growth and development of higher plants.     

Polycomb‐group histone methyltransferase CLF is required for proper somatic recombination in Arabidopsis

Homologous recombination (HR) is a key process during meiosis in reproductive cells and the DNA damage repair process in somatic cells. Although chromatin structure is thought to be crucial for HR, only a small number of chromatin modifiers have been studied in HR regulation so far. Here, we investigated the function of CURLY LEAF (CLF), a Polycomb‐group (PcG) gene responsible for histone3 lysine 27 trimethylation (H3K27me3), in somatic and meiotic HR in Arabidopsis thaliana. Although fluorescent protein reporter assays in pollen and seeds showed that the frequency of meiotic cross‐over in the loss‐of‐function mutant clf‐29 was not significantly different from that in wild type, there was a lower frequency of HR in clf‐29 than in wild type under normal conditions and under bleomycin treatment. The DNA damage levels were comparable between clf‐29 and wild type, even though several DNA damage repair genes (e.g. ATM, BRCA2a, RAD50, RAD51, RAD54, and PARP2) were expressed at lower levels in clf‐29. Under bleomycin treatment, the expression levels of DNA repair genes were similar in clf‐29 and wild type, thus CLF may also regulate HR via other mechanisms. These findings expand the current knowledge of PcG function and contribute to general interests of epigenetic regulation in genome stability regulation.     

Non‐specific phospholipases C,NPC2 and NPC6, are required for root growth in Arabidopsis

Mutants in lipid metabolism often show a lethal phenotype during reproduction that prevents investigating a specific role of the lipid during different developmental processes. We focused on two non‐specific phospholipases C, NPC2 and NPC6, whose double knock‐out causes a gametophyte‐lethal phenotype. To investigate the role of NPC2 and NPC6 during vegetative growth, we produced transgenic knock‐down mutant lines that circumvent the lethal effect during gametogenesis. Despite no defect observed in leaves, root growth was significantly retarded, with abnormal cellular architecture in root columella cells. Furthermore, the short root phenotype was rescued by exogenous supplementation of phosphocholine, a product of non‐specific phospholipase C (NPC) ‐catalyzed phosphatidylcholine hydrolysis. The expression of phospho‐base N‐methyltransferase 1 (PMT1), which produces phosphocholine and is required for root growth, was induced in the knock‐down mutant lines and was attenuated after phosphocholine supplementation. These results suggest that NPC2 and NPC6 may be involved in root growth by producing phosphocholine via metabolic interaction with a PMT‐catalyzed pathway, which highlights a tissue‐specific role of NPC enzymes in vegetative growth beyond the gametophyte‐lethal phenotype.     

Ethylene mediates dichromate‐induced inhibition of primary root growth by altering AUX1 expression and auxin accumulation in Arabidopsis thaliana

The hexavalent form of chromium [Cr(VI)] causes a major reduction in yield and quality of crops worldwide. The root is the first plant organ that interacts with Cr(VI) toxicity, which inhibits primary root elongation, but the underlying mechanisms of this inhibition remain elusive. In this study, we investigate the possibility that Cr(VI) reduces primary root growth of Arabidopsis by modulating the cell cycle‐related genes and that ethylene signalling contributes to this process. We show that Cr(VI)‐mediated inhibition of primary root elongation was alleviated by the ethylene perception and biosynthesis antagonists silver and cobalt, respectively. Furthermore, the ethylene signalling defective mutants (ein2‐1 and etr1‐3) were insensitive, whereas the overproducer mutant (eto1‐1) was hypersensitive to Cr(VI). We also report that high levels of Cr(VI) significantly induce the distribution and accumulation of auxin in the primary root tips, but this increase was significantly suppressed in seedlings exposed to silver or cobalt. In addition, genetic and physiological investigations show that AUXIN‐RESISTANT1 (AUX1) participates in Cr(VI)‐induced inhibition of primary root growth. Taken together, our results indicate that ethylene mediates Cr(VI)‐induced inhibition of primary root elongation by increasing auxin accumulation and polar transport by stimulating the expression of AUX1.     

The cohesin complex is required for the DNA damage‐induced G2/M checkpoint in mammalian cells

Cohesin complexes mediate sister chromatid cohesion. Cohesin also becomes enriched at DNA double‐strand break sites and facilitates recombinational DNA repair. Here, we report that cohesin is essential for the DNA damage‐induced G2/M checkpoint. In contrast to cohesin's role in DNA repair, the checkpoint function of cohesin is independent of its ability to mediate cohesion. After RNAi‐mediated depletion of cohesin, cells fail to properly activate the checkpoint kinase Chk2 and have defects in recruiting the mediator protein 53BP1 to DNA damage sites. Earlier work has shown that phosphorylation of the cohesin subunits Smc1 and Smc3 is required for the intra‐S checkpoint, but Smc1/Smc3 are also subunits of a distinct recombination complex, RC‐1. It was, therefore, unknown whether Smc1/Smc3 function in the intra‐S checkpoint as part of cohesin. We show that Smc1/Smc3 are phosphorylated as part of cohesin and that cohesin is required for the intra‐S checkpoint. We propose that accumulation of cohesin at DNA break sites is not only needed to mediate DNA repair, but also facilitates the recruitment of checkpoint proteins, which activate the intra‐S and G2/M checkpoints.     

Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion

ABCG11/WBC11, an ATP binding cassette (ABC) transporter from Arabidopsis thaliana, is a key component of the export pathway for cuticular lipids. Arabidopsis wbc11 T-DNA insertional knock-out mutants exhibited lipidic inclusions inside epidermal cells similar to the previously characterized wax transporter mutant cer5, with a similar strong reduction in the alkanes of surface waxes. Moreover, the wbc11 knock-out mutants also showed defects not present in cer5, including post-genital organ fusions, stunted growth and a reduction in cutin load on the plant surface. A mutant line previously isolated in a forward genetics screen, called permeable leaves 1 (pel1), was identified as an allele of ABCG11/WBC11. The double knock-out wbc11 cer5 exhibited the same morphological and biochemical phenotypes as the wbc11 knock-out. A YFP-WBC11 fusion protein rescued a T-DNA knock-out mutant and was localized to the plasma membrane. These results show that WBC11 functions in secretion of surface waxes, possibly by interacting with CER5. However, unlike ABCG12/CER5, ABCG11/WBC11 is important to the normal process of cutin formation.     

Enhanced root growth in phosphate‐starved Arabidopsis by stimulating de novo phospholipid biosynthesis through the overexpression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 (LPAT2)

Upon phosphate starvation, plants retard shoot growth but promote root development presumably to enhance phosphate assimilation from the ground. Membrane lipid remodelling is a metabolic adaptation that replaces membrane phospholipids by non‐phosphorous galactolipids, thereby allowing plants to obtain scarce phosphate yet maintain the membrane structure. However, stoichiometry of this phospholipid‐to‐galactolipid conversion may not account for the massive demand of membrane lipids that enables active growth of roots under phosphate starvation, thereby suggesting the involvement of de novo phospholipid biosynthesis, which is not represented in the current model. We overexpressed an endoplasmic reticulum‐localized lysophosphatidic acid acyltransferase, LPAT2, a key enzyme that catalyses the last step of de novo phospholipid biosynthesis. Two independent LPAT2 overexpression lines showed no visible phenotype under normal conditions but showed increased root length under phosphate starvation, with no effect on phosphate starvation response including marker gene expression, root hair development and anthocyanin accumulation. Accompanying membrane glycerolipid profiling of LPAT2‐overexpressing plants revealed an increased content of major phospholipid classes and distinct responses to phosphate starvation between shoot and root. The findings propose a revised model of membrane lipid remodelling, in which de novo phospholipid biosynthesis mediated by LPAT2 contributes significantly to root development under phosphate starvation.