The Arabidopsis SMO2, a homologue of yeast TRM112, modulates progression of cell division during organ growth

Cell proliferation is integrated into developmental progression in multicellular organisms, including plants, and the regulation of cell division is of pivotal importance for plant growth and development. Here, we report the identification of an Arabidopsis SMALL ORGAN 2 (SMO2) gene that functions in regulation of the progression of cell division during organ growth. The smo2 knockout mutant displays reduced size of aerial organs and shortened roots, due to the decreased number of cells in these organs. Further analyses reveal that disruption of SMO2 does not alter the developmental timing but reduces the rate of cell production during leaf and root growth. Moreover, smo2 plants exhibit a constitutive activation of cell cycle‐related genes and over‐accumulation of cells expressing CYCB1;1:β‐glucuronidase (CYCB1;1:GUS) during organogenesis, suggesting that smo2 has a defect in G₂–M phase progression in the cell cycle. SMO2 encodes a functional homologue of yeast TRM112, a plurifunctional component involved in a few cellular events, including tRNA and protein methylation. In addition, the mutation of SMO2 does not appear to affect endoreduplication in Arabidopsis leaf cells. Taken together we postulate that Arabidopsis SMO2 is a conserved yeast TRM112 homologue and SMO2‐mediated cellular events are required for proper progression of cell division in plant growth and development.     

A T-DNA mutation in the RNA helicase eIF4A confers a dose-dependent dwarfing phenotype in Brachypodium distachyon

In a survey of the BrachyTAG mutant population of Brachypodium distachyon, we identified a line carrying a T-DNA insertion in one of the two eukaryotic initiation factor 4A (eIF4A) genes present in the nuclear genome. The eif4a homozygous mutant plants were slow-growing, and exhibited reduced final plant stature due to a decrease in both cell number and cell size, consistent with roles for eIF4A in both cell division and cell growth. Hemizygous plants displayed a semi-dwarfing phenotype, in which stem length was reduced but leaf length was normal. Linkage between the insertion site and phenotype was confirmed, and we show that the level of eIF4A protein is strongly reduced in the mutant. Transformation of the Brachypodium homozygous mutant with a genomic copy of the Arabidopsis eIF4A-1 gene partially complemented the growth phenotype, indicating that gene function is conserved between mono- and dicotyledonous species. This study identifies eIF4A as a novel dose-dependent regulator of stem elongation, and demonstrates the utility of Brachypodium as a model for grass and cereals research.     

mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E

The mammalian target of rapamycin (mTOR) integrates nutrient and mitogen signals to regulate cell growth (increased cell mass and cell size) and cell division. The immunosuppressive drug rapamycin inhibits cell cycle progression via inhibition of mTOR; however, the signaling pathways by which mTOR regulates cell cycle progression have remained poorly defined. Here we demonstrate that restoration of mTOR signaling (by using a rapamycin-resistant mutant of mTOR) rescues rapamycin-inhibited G(1)-phase progression, and restoration of signaling along the mTOR-dependent S6K1 or 4E-BP1/eukaryotic translation initiation factor 4E (eIF4E) pathways provides partial rescue. Furthermore, interfering RNA-mediated reduction of S6K1 expression or overexpression of mTOR-insensitive 4E-BP1 isoforms that block eIF4E activity inhibit G(1)-phase progression individually and additively. Thus, the activities of both the S6K1 and 4E-BP1/eIF4E pathways are required for and independently mediate mTOR-dependent G(1)-phase progression. In addition, overexpression of constitutively active mutants of S6K1 or wild-type eIF4E accelerates serum-stimulated G(1)-phase progression, and stable expression of wild-type S6K1 confers a proliferative advantage in low-serum-containing media, suggesting that the activity of each of these pathways is limiting for cell proliferation. These data demonstrate that, as for the regulation of cell growth and cell size, the S6K1 and 4E-BP1/eIF4E pathways each represent critical mediators of mTOR-dependent cell cycle control.     

BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis

Arabidopsis thaliana brassinosteroid signaling kinases (BSKs) constitute a receptor‐like cytoplasmic kinase sub‐family (RLCK‐XII) with 12 members. Previous analysis demonstrated a positive role for BSK1 and BSK3 in the initial steps of brassinosteroid (BR) signal transduction. To investigate the function of BSKs in plant growth and BR signaling, we characterized T‐DNA insertion lines for eight BSK genes (BSK1–BSK8) and multiple mutant combinations. Simultaneous elimination of three BSK genes caused alterations in growth and the BR response, and the most severe phenotypes were observed in the bsk3,4,7,8 quadruple and bsk3,4,6,7,8 pentuple mutants, which displayed reduced rosette size, leaf curling and enhanced leaf inclination. In addition, upon treatment with 24‐epibrassinolide, these mutants showed reduced hypocotyl elongation, enhanced root growth and alteration in the expression of BR‐responsive genes. Some mutant combinations also showed antagonistic interactions. In support of a redundant function in BR signaling, multiple BSKs interacted in vivo with the BR receptor BRI1, and served as its phosphorylation substrates in vitro. The BIN2 and BIL2 GSK3‐like kinases, which are negative regulators of BR signaling, interacted in vivo with BSKs and phosphorylated them in vitro, probably at different sites to BRI1. This study demonstrates redundant biological functions for BSKs, and suggests the existence of a regulatory link between BSKs and GSK3‐like kinases.     

Development of broad virus resistance in non‐transgenic cucumber using CRISPR/Cas9 technology

Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N′ and C′ termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off‐target sites. Non‐transgenic heterozygous eif4e mutant plants were selected for the production of non‐transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus‐W. In contrast, heterozygous mutant and non‐mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non‐transgenically, not visibly affecting plant development and without long‐term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.     

Rice DECUSSATE controls phyllotaxy by affecting the cytokinin signaling pathway

Phyllotaxy is defined as the spatial arrangement of leaves on the stem. The mechanism responsible for this extremely regular pattern is one of the most fascinating enigmas in plant biology. In this study, we identified a gene regulating the phyllotactic pattern in rice. Loss‐of‐function mutants of the DECUSSATE (DEC) gene displayed a phyllotactic conversion from normal distichous pattern to decussate. The dec mutants had an enlarged shoot apical meristem with enhanced cell division activity. In contrast to the shoot apical meristem, the size of the root apical meristem in the dec mutants was reduced, and cell division activity was suppressed. These phenotypes indicate that DEC has opposite functions in the shoot apical meristem and root apical meristem. Map‐based cloning revealed that DEC encodes a plant‐specific protein containing a glutamine‐rich region and a conserved motif. Although its molecular function is unclear, the conserved domain is shared with fungi and animals. Expression analysis showed that several type A response regulator genes that act in the cytokinin signaling pathway were down‐regulated in the dec mutant. In addition, dec seedlings showed a reduced responsiveness to exogenous cytokinin. Our results suggest that DEC controls the phyllotactic pattern by affecting cytokinin signaling in rice.     

The Arabidopsis eukaryotic translation initiation factor 3, subunit F (AteIF3f), is required for pollen germination and embryogenesis

Previous studies have shown that subunits E (eIF3e), F (eIF3f) and H (elF3h) of eukaryotic translation initiation factor 3 play important roles in cell development in humans and yeast. eIF3e and eIF3h have also been reported to be important for normal cell growth in Arabidopsis. However, the functions of subunit eIF3f remain largely unknown in plant species. Here we report characterization of mutants for the Arabidopsis eIF3f (AteIF3f) gene. AteIF3f encodes a protein that is highly expressed in pollen grains, developing embryos and root tips, and interacts with Arabidopsis eIF3e and eIF3h proteins. A Ds insertional mutation in AteIF3f disrupted pollen germination and embryo development. Expression of some of the genes that are essential for pollen tube growth and embryogenesis is down‐regulated in ateif3f‐1 homozygous seedlings obtained by pollen rescue. These results suggested that AteIF3f might play important roles in Arabidopsis cell growth and differentiation in combination with eIF3e and eIF3h.     

Characterization of plant eukaryotic translation initiation factor 6 (eIF6) genes: The essential role in embryogenesis and their differential expression in Arabidopsis and rice

Eukaryotic translation initiation factor 6 (eIF6) is an essential component of ribosome biogenesis. In our present study, we characterize plant eIF6 genes for the first time. Although a single gene encodes eIF6 in yeast and animals, two genes were found to encode proteins homologous to animal and yeast eIF6 in Arabidopsis and rice, denoted At-eIF6;1 and At-eIF6;2, and Os-eIF6;1 and Os-eIF6;2, respectively. Analysis of the yeast eif6 (tif6) mutant suggested that plant eIF6, at least in the case of At-eIF6;1, can complement the essential function of eIF6 in yeast. Evidence for the essential role of eIF6 in plants was also provided by the embryonic-lethal phenotype of the at-eif6;1 mutant. In contrast, At-eIF6;2 appears not to be essential due to its very low expression level and the normal growth phenotype of the eif6;2 mutants. Consistent with the putative role of plant eIF6 in ribosome biogenesis, At-eIF6;1 is predominately expressed in tissues where cell division actively proceeds under the control of intronic cis-regulatory elements. On the other hand, both Os-eIF6;1 and Os-eIF6;2 are probably active genes because they are expressed at significant expression levels. Interestingly, the supply of ammonium nitrate as a plant nutrient was found to induce specifically the expression of Os-eIF6;2. Our present findings indicate that the eIF6 genes have differently evolved in plant and animal kingdoms and also in distinct plant species.     

Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. Two genes, TIF51A and TIF51B, encode eIF5A in the yeast Saccharomyces cerevisiae. In an effort to understand the structure–function relationship of eIF5A, we have generated yeast mutants by introducing plasmid-borne tif51A into a double null strain where both TIF51A and TIF51B have been disrupted. One of the mutants, tsL102A strain (tif51A L102A tif51aΔ tif51bΔ) exhibits a strong temperature-sensitive growth phenotype. At the restrictive temperature, tsL102A strain also exhibits a cell shape change, a lack of volume change in response to temperature increase and becomes more sensitive to ethanol, a hallmark of defects in the PKC/WSC cell wall integrity pathway. In addition, a striking change in actin dynamics and a complete cell cycle arrest at G1 phase occur in tsL102A cells at restrictive temperature. The temperature-sensitivity of tsL102A strain is due to a rapid loss of mutant eIF5A with the half-life reduced from 6 h at permissive temperature to 20 min at restrictive temperature. Phenylmethyl sulfonylfluoride (PMSF), an irreversible inhibitor of serine protease, inhibited the degradation of mutant eIF5A and suppressed the temperature-sensitive growth arrest. Sorbitol, an osmotic stabilizer that complement defects in PKC/WSC pathways, stabilizes the mutant eIF5A and suppresses all the observed temperature-sensitive phenotypes.     

OsCYP2, a chaperone involved in degradation of auxin‐responsive proteins,plays crucial roles in rice lateral root initiation

Auxin plays a pivotal role in many facets of plant development. It acts by inducing the interaction between auxin‐responsive [auxin (AUX)/indole‐3‐acetic acid (IAA)] proteins and the ubiquitin protein ligase SCF^TIR to promote the degradation of the AUX/IAA proteins. Other cofactors and chaperones that participate in auxin signaling remain to be identified. Here, we characterized rice (Oryza sativa) plants with mutations in a cyclophilin gene (OsCYP2). cyp2 mutants showed defects in auxin responses and exhibited a variety of auxin‐related growth defects in the root. In cyp2 mutants, lateral root initiation was blocked after nuclear migration but before the first anticlinal division of the pericycle cell. Yeast two‐hybrid and in vitro pull‐down results revealed an association between OsCYP2 and the co‐chaperone Suppressor of G2 allele of skp1 (OsSGT1). Luciferase complementation imaging assays further supported this interaction. Similar to previous findings in an Arabidopsis thaliana SGT1 mutant (atsgt1b), degradation of AUX/IAA proteins was retarded in cyp2 mutants treated with exogenous 1‐naphthylacetic acid. Our results suggest that OsCYP2 participates in auxin signal transduction by interacting with OsSGT1.     

A replication stress-induced synchronization method for Arabidopsis thaliana root meristems

Synchronized cell cultures are an indispensable tool for the identification and understanding of key regulators of the cell cycle. Nevertheless, the use of cell cultures has its disadvantages, because it represents an artificial system that does not completely mimic the endogenous conditions that occur in organized meristems. Here, we present a new and easy method for Arabidopsis thaliana root tip synchronization by hydroxyurea treatment. A major advantage of the method is the possibility of investigating available Arabidopsis cell‐cycle mutants without the need to generate cell cultures. As a proof of concept, the effects of over‐expression of a dominant negative allele of the B‐type cyclin‐dependent kinase CDKB1;1 gene on cell‐cycle progression were tested. The previously observed prolonged G₂ phase was confirmed, but was found to be compensated for by a reduced G₁ phase. Furthermore, altered S‐phase kinetics indicated a functional role for CDKB1;1 during the replication process.     

Translational control of lipogenic enzymes in the cell cycle of synchronous,growing yeast cells

Translational control during cell division determines when cells start a new cell cycle, how fast they complete it, the number of successive divisions, and how cells coordinate proliferation with available nutrients. The translational efficiencies of mRNAs in cells progressing synchronously through the mitotic cell cycle, while preserving the coupling of cell division with cell growth, remain uninvestigated. We now report comprehensive ribosome profiling of a yeast cell size series from the time of cell birth, to identify mRNAs under periodic translational control. The data reveal coordinate translational activation of mRNAs encoding lipogenic enzymes late in the cell cycle including Acc1p, the rate‐limiting enzyme acetyl‐CoA carboxylase. An upstream open reading frame (uORF) confers the translational control of ACC1 and adjusts Acc1p protein levels in different nutrients. The ACC1 uORF is relevant for cell division because its ablation delays cell cycle progression, reduces cell size, and suppresses the replicative longevity of cells lacking the Sch9p protein kinase regulator of ribosome biogenesis. These findings establish an unexpected relationship between lipogenesis and protein synthesis in mitotic cell divisions.     

A double‐mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions

Mitogen‐activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single‐mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double‐mutants are created from a large library of single‐mutant lines. Here we describe a new collection of 275 double‐mutant lines derived from a library of single‐mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high‐throughput double‐mutant generating pipeline using a system for growing Arabidopsis seedlings in 96‐well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double‐mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single‐mutant line. Seeds for this double‐mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double‐mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling.     

A dual‐color marker system for in vivo visualization of cell cycle progression in Arabidopsis

Visualization of the spatiotemporal pattern of cell division is crucial to understand how multicellular organisms develop and how they modify their growth in response to varying environmental conditions. The mitotic cell cycle consists of four phases: S (DNA replication), M (mitosis and cytokinesis), and the intervening G1 and G2 phases; however, only G2/M‐specific markers are currently available in plants, making it difficult to measure cell cycle duration and to analyze changes in cell cycle progression in living tissues. Here, we developed another cell cycle marker that labels S‐phase cells by manipulating Arabidopsis CDT1a, which functions in DNA replication origin licensing. Truncations of the CDT1a coding sequence revealed that its carboxy‐terminal region is responsible for proteasome‐mediated degradation at late G2 or in early mitosis. We therefore expressed this region as a red fluorescent protein fusion protein under the S‐specific promoter of a histone 3.1‐type gene, HISTONE THREE RELATED2 (HTR2), to generate an S/G2 marker. Combining this marker with the G2/M‐specific CYCB1‐GFP marker enabled us to visualize both S to G2 and G2 to M cell cycle stages, and thus yielded an essential tool for time‐lapse imaging of cell cycle progression. The resultant dual‐color marker system, Cell Cycle Tracking in Plant Cells (Cytrap), also allowed us to identify root cells in the last mitotic cell cycle before they entered the endocycle. Our results demonstrate that Cytrap is a powerful tool for in vivo monitoring of the plant cell cycle, and thus for deepening our understanding of cell cycle regulation in particular cell types during organ development.     

Expression of TaCYP78A3, a gene encoding cytochrome P450 CYP78A3 protein in wheat (Triticum aestivum L.), affects seed size

Several studies have described quantitative trait loci (QTL) for seed size in wheat, but the relevant genes and molecular mechanisms remain largely unknown. Here we report the functional characterization of the wheat TaCYP78A3 gene and its effect on seed size. TaCYP78A3 encoded wheat cytochrome P450 CYP78A3, and was specifically expressed in wheat reproductive organs. TaCYP78A3 activity was positively correlated with the final seed size. Its silencing caused a reduction of cell number in the seed coat, resulting in an 11% decrease in wheat seed size, whereas TaCYP78A3 over‐expression induced production of more cells in the seed coat, leading to an 11–48% increase in Arabidopsis seed size. In addition, the cell number in the final seed coat was determined by the TaCYP78A3 expression level, which affected the extent of integument cell proliferation in the developing ovule and seed. Unfortunately, TaCYP78A3 over‐expression in Arabidopsis caused a reduced seed set due to an ovule developmental defect. Moreover, TaCYP78A3 over‐expression affected embryo development by promoting embryo integument cell proliferation during seed development, which also ultimately affected the final seed size in Arabidopsis. In summary, our results indicated that TaCYP78A3 plays critical roles in influencing seed size by affecting the extent of integument cell proliferation. The present study provides direct evidence that TaCYP78A3 affects seed size in wheat, and contributes to an understanding of the cellular basis of the gene influencing seed development.