MOL1 is required for cambium homeostasis in Arabidopsis

Plants maintain pools of pluripotent stem cells which allow them to constantly produce new tissues and organs. Stem cell homeostasis in shoot and root tips depends on negative regulation by ligand–receptor pairs of the CLE peptide and leucine‐rich repeat receptor‐like kinase (LRR‐RLK) families. However, regulation of the cambium, the stem cell niche required for lateral growth of shoots and roots, is poorly characterized. Here we show that the LRR‐RLK MOL1 is necessary for cambium homeostasis in Arabidopsis thaliana. By employing promoter reporter lines, we reveal that MOL1 is active in a domain that is distinct from the domain of the positively acting CLE41/PXY signaling module. In particular, we show that MOL1 acts in an opposing manner to the CLE41/PXY module and that changing the domain or level of MOL1 expression both result in disturbed cambium organization. Underlining discrete roles of MOL1 and PXY, both LRR‐RLKs are not able to replace each other when their expression domains are interchanged. Furthermore, MOL1 but not PXY is able to rescue CLV1 deficiency in the shoot apical meristem. By identifying genes mis‐expressed in mol1 mutants, we demonstrate that MOL1 represses genes associated with stress‐related ethylene and jasmonic acid hormone signaling pathways which have known roles in coordinating lateral growth of the Arabidopsis stem. Our findings provide evidence that common regulatory mechanisms in different plant stem cell niches are adapted to specific niche anatomies and emphasize the importance of a complex spatial organization of intercellular signaling cascades for a strictly bidirectional tissue production.     

DbpA is a region‐specific RNA helicase

DbpA is a DEAD‐box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA‐like catalytic core responsible for double‐helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core's grasp. In other words, it is not known if DbpA is a site‐specific enzyme or region‐specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region‐specific or a site‐specific enzyme. Our data suggest that DbpA is a region‐specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C‐terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double‐helices in its vicinity. The only requirement for a double‐helix to serve as a DbpA substrate is for the double‐helix to be positioned within the catalytic core's grasp.     

The DEAD box RNA helicase VBH-1 is required for germ cell function in C. elegans

Vasa and Belle are conserved DEAD box RNA helicases required for germ cell function. Homologs of this group of proteins in several species, including mammals, are able to complement a mutation in yeast (DED1) suggesting that their function is highly conserved. It has been proposed that these proteins are required for mRNA translation regulation, but their specific mechanism of action is still unknown. Here we describe functions of VBH-1, a C. elegans protein closely related to Belle and Vasa. VBH-1 is expressed specifically in the C. elegans germline, where it is associated with P granules, the C. elegans germ plasm counterpart. vbh-1(RNAi) animals produce fewer offspring than wild type because of defects in oocyte and sperm production, and embryonic lethality. We also find that VBH-1 participates in the sperm/oocyte switch in the hermaphrodite gonad. We conclude that VBH-1 and its orthologs may perform conserved roles in fertility and development.     

CLUMSY VEIN,the Arabidopsis DEAH‐box Prp16 ortholog,is required for auxin‐mediated development

Pre‐messenger RNA (pre‐mRNA) splicing is essential in eukaryotic cells. In animals and yeasts, the DEAH‐box RNA‐dependent ATPase Prp16 mediates conformational change of the spliceosome, thereby facilitating pre‐mRNA splicing. In yeasts, Prp16 also plays an important role in splicing fidelity. Conversely, PRP16 orthologs in Chlamydomonas reinhardtii and nematode do not have an important role in general pre‐mRNA splicing, but are required for gene silencing and sex determination, respectively. Functions of PRP16 orthologs in higher plants have not been described until now. Here we show that the CLUMSY VEIN (CUV) gene encoding the unique Prp16 ortholog in Arabidopsis thaliana facilitates auxin‐mediated development including male‐gametophyte transmission, apical–basal patterning of embryonic and gynoecium development, stamen development, phyllotactic flower positioning, and vascular development. cuv‐1 mutation differentially affects splicing and expression of genes involved in auxin biosynthesis, polar auxin transport, auxin perception and auxin signaling. The cuv‐1 mutation does not have an equal influence on pre‐mRNA substrates. We propose that Arabidopsis PRP16/CUV differentially facilitates expression of genes, which include genes involved in auxin biosynthesis, transport, perception and signaling, thereby collectively influencing auxin‐mediated development.     

The Arabidopsis heterotrimeric G‐protein β subunit,AGB1, is required for guard cell calcium sensing and calcium‐induced calcium release

Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Ca_o responses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cyt oscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double‐mutants, as well as those of the agg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast with ABA‐regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6‐detected [Ca²⁺]_cyt oscillations in response to Ca_o, initiating only a single [Ca²⁺]_cyt spike. Experimentally imposed [Ca²⁺]_cyt oscillations restored stomatal closure in agb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Ca_o induced InsP3 production in Col but not in agb1. In sum, G‐protein signaling via AGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_o apparently require Ca²⁺‐induced Ca²⁺ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.     

The Arabidopsis STRESS RESPONSE SUPPRESSOR DEAD‐box RNA helicases are nucleolar‐ and chromocenter‐localized proteins that undergo stress‐mediated relocalization and are involved in epigenetic gene silencing

DEAD‐box RNA helicases are involved in many aspects of RNA metabolism and in diverse biological processes in plants. Arabidopsis thaliana mutants of two DEAD‐box RNA helicases, STRESS RESPONSE SUPPRESSOR1 (STRS1) and STRS2 were previously shown to exhibit tolerance to abiotic stresses and up‐regulated stress‐responsive gene expression. Here, we show that Arabidopsis STRS‐overexpressing lines displayed a less tolerant phenotype and reduced expression of stress‐induced genes confirming the STRSs as attenuators of Arabidopsis stress responses. GFP–STRS fusion proteins exhibited localization to the nucleolus, nucleoplasm and chromocenters and exhibited relocalization in response to abscisic acid (ABA) treatment and various stresses. This relocalization was reversed when stress treatments were removed. The STRS proteins displayed mis‐localization in specific gene‐silencing mutants and exhibited RNA‐dependent ATPase and RNA‐unwinding activities. In particular, STRS2 showed mis‐localization in three out of four mutants of the RNA‐directed DNA methylation (RdDM) pathway while STRS1 was mis‐localized in the hd2c mutant that is defective in histone deacetylase activity. Furthermore, heterochromatic RdDM target loci displayed reduced DNA methylation and increased expression in the strs mutants. Taken together, our findings suggest that the STRS proteins are involved in epigenetic silencing of gene expression to bring about suppression of the Arabidopsis stress response.     

Arabidopsis OTU1, a linkage‐specific deubiquitinase,is required for endoplasmic reticulum‐associated protein degradation

Endoplasmic reticulum (ER)‐associated degradation (ERAD) is part of the ER protein quality‐control system (ERQC), which is critical for the conformation fidelity of most secretory and membrane proteins in eukaryotic organisms. ERAD is thought to operate in plants with core machineries highly conserved to those in human and yeast; however, little is known about the plant ERAD system. Here we report the characterization of a close homolog of human OTUB1 in Arabidopsis thaliana, designated as AtOTU1. AtOTU1 selectively hydrolyzes several types of ubiquitin chains and these activities depend on its conserved protease domain and/or the unique N‐terminus. The otu1 null mutant is sensitive to high salinity stress, and particularly agents that cause protein misfolding. It turns out that AtOTU1 is required for the processing of known plant ERAD substrates such as barley powdery mildew O (MLO) alleles by virtue of its association with the CDC48 complex through its N‐terminal region. These observations collectively define AtOTU1 as an OTU domain‐containing deubiquitinase involved in Arabidopsis ERAD.     

Arabidopsis replication factor C4 is critical for DNA replication during the mitotic cell cycle

Replication factor C (RFC) is a conserved eukaryotic complex consisting of RFC1/2/3/4/5. It plays important roles in DNA replication and the cell cycle in yeast and fruit fly. However, it is not very clear how RFC subunits function in higher plants, except for the Arabidopsis (At) subunits AtRFC1 and AtRFC3. In this study, we investigated the functions of AtRFC4 and found that loss of function of AtRFC4 led to an early sporophyte lethality that initiated as early as the elongated zygote stage, all defective embryos arrested at the two‐ to four‐cell embryo proper stage, and the endosperm possessed six to eight free nuclei. Complementation of rfc4‐1/+ with AtRFC4 expression driven through the embryo‐specific DD45pro and ABI3pro or the endosperm‐specific FIS2pro could not completely restore the defective embryo or endosperm, whereas a combination of these three promoters in rfc4‐1/+ enabled the aborted ovules to develop into viable seeds. This suggests that AtRFC4 functions simultaneously in endosperm and embryo and that the proliferation of endosperm is critical for embryo maturation. Assays of DNA content in rfc4‐1/+ verified that DNA replication was disrupted in endosperm and embryo, resulting in blocked mitosis. Moreover, we observed a decreased proportion of late S‐phase and M‐phase cells in the rfc4‐1/–^{FIS2;DD45;ABI3pro::AtRFC4} seedlings, suggesting that incomplete DNA replication triggered cell cycle arrest in cells of the root apical meristem. Therefore, we conclude that AtRFC4 is a crucial gene for DNA replication.     

TRANSPARENT TESTA 13 is a tonoplast P3A‐ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P‐type H⁺‐ATPases in the plasma membrane, and multimeric vacuolar‐type H⁺‐ATPases (V‐ATPases) and vacuolar H⁺‐pyrophosphatases (H⁺‐PPases) in endomembranes. Here, we show that reduced accumulation of proanthocyanidins (PAs) and hence the diminished brown seed coloration found in the Arabidopsis thaliana mutant transparent testa 13 (tt13) is caused by disruption of the gene encoding the P_3A‐ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants. Cells of the tt13 seed coat endothelium do not contain PA‐filled central vacuoles as observed in the wild‐type. tt13 phenocopies tt12, a mutant that is defective in vacuolar import of the PA precursor epicatechin. Our data show that vacuolar loading with PA precursors depends on TT13. Consistent with the tt13 phenotype, but in contrast to other isoforms of P‐type H⁺‐ATPases, TT13 localizes to the tonoplast. PA accumulation in tt13 is partially restored by expression of the tonoplast localized H⁺‐PPase VHP1. Our findings indicate that the P_3A‐ATPase TT13 functions as a proton pump in the tonoplast of seed coat endothelium cells, and generates the driving force for TT12‐mediated transport of PA precursors to the vacuole.     

Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing

Dicer proteins are central to the different mechanisms involving RNA interference. Plants have evolved multiple DICER‐LIKE (DCL) copies, thus enabling functional diversification. In Arabidopsis, DCL2 and DCL4 process double‐stranded RNA into 22 and 21 nucleotide small interfering (si)RNAs, respectively, and have overlapping functions with regards to virus and transgene silencing. Nonetheless, some studies have reported that dcl2 or dcl4 single mutations are sometimes sufficient to hinder silencing. To better dissect the role of DCL2 and DCL4, we analyzed silencing kinetics and efficiencies using different transgenic systems in single and double mutant backgrounds. The results indicate that DCL2 stimulates transitivity and secondary siRNA production through DCL4 while being sufficient for silencing on its own. Notably, silencing of 35S‐driven transgenes functions more efficiently in dcl4 mutants, indicating that DCL4 mostly obscures DCL2 in wild‐type plants. Nonetheless, in a dcl4 mutant compromised in phloem‐originating silencing, ectopically expressed DCL2 allows restoration of silencing, suggesting that DCL2 is not, or poorly, expressed in phloem. Remarkably, this ectopic DCL2 contribution to phloem‐originating silencing is dependent on the activity of RNA‐DEPENDENT RNA POLYMERASE6. These results indicate that, despite differences in the silencing activity of their small RNA products, DCL2 and DCL4 mostly act redundantly yet hierarchically when present simultaneously.     

Cyclin‐dependent kinase‐activating kinases CDKD;1 and CDKD;3 are essential for preserving mitotic activity in Arabidopsis thaliana

For the full activation of cyclin‐dependent kinases (CDKs), not only cyclin binding but also CDK phosphorylation is required. This activating phosphorylation is mediated by CDK‐activating kinases (CAKs). Arabidopsis has four genes showing similarity to vertebrate‐type CAKs, three CDKDs (CDKD;1‐CDKD;3) and one CDKF (CDKF;1). We previously found that the cdkf;1 mutant is defective in post‐embryonic development, even though the kinase activities of core CDKs remain unchanged relative to the wild type. This raised a question about the involvement of CDKDs in CDK activation in planta. Here we report that the cdkd;1 cdkd;3 double mutant showed gametophytic lethality. Most cdkd;1‐1 cdkd;3‐1 pollen grains were defective in pollen mitosis I and II, producing one‐cell or two‐cell pollen grains that lacked fertilization ability. We also found that the double knock‐out of CDKD;1 and CDKD;3 caused arrest and/or delay in the progression of female gametogenesis at multiple steps. Our genetic analyses revealed that the functions of CDKF;1 and CDKD;1 or CDKD;3 do not overlap, either during gametophyte and embryo development or in post‐embryonic development. Consistent with these analyses, CDKF;1 expression in the cdkd;1‐1 cdkd;3‐1 mutant could not rescue the gametophytic lethality. These results suggest that, in Arabidopsis, CDKD;1 and CDKD;3 function as CAKs controlling mitosis, whereas CDKF;1 plays a distinct role, mainly in post‐embryonic development. We propose that CDKD;1 and CDKD;3 phosphorylate and activate all core CDKs, CDKA, CDKB1 and CDKB2, thereby governing cell cycle progression throughout plant development.     

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.