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Fruit shape is an important biological trait that is also of special commercial value in tomato. The SUN gene has been known as a key regulator of tomato fruit elongation for years, but the molecular mechanisms underlying its transcriptional regulation remain little understood. Here, a unique BZR1-like transcription factor, BZR1.7, was identified as a trans-acting factor of the SUN gene promoter that bound to the conserved E-box of the promoter to promote SUN gene expression. Overexpression of BZR1.7 in tomato led to elevated SUN gene expression and formation of elongated fruits. Plants of the BZR1.7 knockout mutant created by gene editing did not exhibit an observable fruit shape phenotype, suggesting possible functional redundancy of BZR1-like genes in tomato. There were seven BZR1-like genes in the tomato genome and overexpression of BZR1.5 and BZR1.6 led to elongated fruit phenotypes similar to those observed in the BZR1.7 overexpression lines, further supporting the notion of functional redundancy of BZR1-like genes in tomato fruit shape specification. Microscopic analysis revealed that there was a decreased number of cell layers in the fruit pericarp in the BZR1.7 overexpression lines. These findings offer new insights into the regulatory mechanism by which BZR1.7 promotes SUN gene expression and regulates fruit elongation in tomato.

Fertilization is a key event for sexually reproducing plants. Pollen–stigma adhesion, which is the first step in male–female interaction during fertilization, requires proper pollen wall patterning. Callose, which is a β-1.3-glucan, is an essential polysaccharide that is required for pollen development and pollen wall formation. Mutations in CALLOSE SYNTHASE 5 (CalS5) disrupt male meiotic callose accumulation; however, how CalS5 activity and callose synthesis are regulated is not fully understood. In this paper, we report the isolation of a kompeito-1 (kom-1) mutant defective in pollen wall patterning and pollen–stigma adhesion in Arabidopsis thaliana. Callose was not accumulated in kom-1 meiocytes or microspores, which was very similar to the cals5 mutant. The KOM gene encoded a member of a subclass of Rhomboid serine protease proteins that lacked active site residues. KOM was localized to the Golgi apparatus, and both KOM and CalS5 genes were highly expressed in meiocytes. A 220 kDa CalS5 protein was detected in wild-type (Col-0) floral buds but was dramatically reduced in kom-1. These results suggested that KOM was required for CalS5 protein accumulation, leading to the regulation of meiocyte-specific callose accumulation and pollen wall formation.

Fruit shape is an important biological trait that is also of special commercial value in tomato. The

The domestication of tomato has led to striking variations in fruit morphology. Here, we show a genome-wide association study (GWAS) to understand the development of the fruit tip and describe aPOINTED TIP(PT) gene that encodes a C2H2-type zinc finger transcription factor. A single nucleotide polymorphism is found to change a histidine (H) to an arginine (R) in the C2H2 domain of PT and the two alleles are referred to asPTHandPTR. Knocking outPTHleads to development of pointed tip fruit.PTHfunctions to suppress pointed tip formation by downregulating the transcription ofFRUTFULL 2(FUL2), which alters the auxin transport. Our evolutionary analysis and previous studies by others suggest that thePTRallele likely hitch-hiked along with other selected loci during the domestication process. This study uncovers variation inPTand molecular mechanism underlying fruit tip development in tomato.

Awns are extending structures from lemmas in grasses and are very active in photosynthesis, contributing directly to the filling of the developing grain. Barley (Hordeum vulgare L.) awns are highly diverse in shape and length and are known to be controlled by multiple awn-related genes. The genetic effects of these genes on awn diversity and development in barley are multiplexed and include complementary effect, cumulative effect, duplicate effect, recessive epistasis, dominant epistasis, and inhibiting effect, each giving a unique modified Mendelian ratio of segregation. The complexity of gene interactions contributes to the awn diversity in barley. Excessive gene interactions create a challenging task for genetic mapping and specific strategies have to be developed for mapping genes with specific interactive effects. Awn gene interactions can occur at different levels of gene expression, from the transcription factor-mediated gene transcription to the regulation of enzymes and metabolic pathways. A better understanding of gene interactions will greatly facilitate deciphering the genetic mechanisms underlying barley awn diversity and development.

Reactive oxygen species (ROS) are inevitably generated in aerobic organisms as by-products of common metabolism and as the result of defense and development. ROS readily oxidizes methionine (Met) residues of proteins to form Met-R-sulfoxide or Met-S-sulfoxide (MetSO), resulting in protein inactivation or malfunction. Although it is known that MetSO can be reverted to Met by methionine sulfoxide reductase (Msr), the mechanism how Msr interacts with its target proteins is poorly understood. In this study, two target proteins of tomato MsrB2 (SlMsrB2), catalase 2 (CAT2) and the Rubisco small subunit RBCS3B, were identified. Silencing of SlMsrB2 by RNA interference (RNAi) in tomato led to decreased drought tolerance, accompanied by increased ROS accumulation and chlorophyll degradation. By contrast, overexpression of SlMsrB2 in tomato significantly reduced ROS accumulation and enhanced drought tolerance. Protein interaction analysis showed that SlMsrB2 interacts with CAT2 and RBCS3B in vitro and in planta. Silencing of CAT2 by RNAi and RBCS3B by virus-induced gene silencing (VIGS) resulted in development of pale green leaves and enhanced ROS accumulation in tomato plants. These results demonstrate that SlMsrB2 functions in drought tolerance and promotes chlorophyll accumulation by modulating ROS accumulation.

Barley (Hordeum vulgare) awns contribute to grain yield, but the genetic basis of awn development remains largely unclear. Five barley lines differing in awn traits and row types were used to create four F2 populations. Genetic analyses revealed that four pairs of genes were involved in awn development: A/a (awnless/awned), B/b (awnless/awned), H/h (hooded/straight), and L/l (long/short). Of these four loci, A, H and L functioned on both central rows (CR) and lateral rows (LR) of the barley spikes, while B exhibited effect only on LR. A and B had duplicate effects on LR, and both showed dominant epistasis to loci H and L, whereas H was epistatic to L. Meanwhile, A and B were found to be genetically linked, with a row-type locus V located between them. The genetic distances of A-V and B-V were estimated to be 9.6 and 7.7 cM, respectively. Literature search suggested that A, H and V may correspond to the reported Lks1, Kap1 and Vrs1, respectively, whereas B is a novel gene specifically controlling awn development on LR, designated as Lsa1 for lateral spikelet awnless 1. The only barley homolog of wheat awn inhibitor gene B1, HORVU2Hr1G077570, is a potential candidate of Lsa1.

The symbiosis receptor kinase SymRK plays an essential role in symbiotic signal transduction and nodule organogenesis. Several proteins bind to SymRK, but how the symbiosis signals are transduced from SymRK to downstream components remains elusive. We previously demonstrated that both SymRK interacting protein 1 (SIP1, an ARID-type DNA-binding protein) and SymRK interacting E3 ligase [SIE3, a RING (Really Interesting New Gene)-containing E3 ligase] interact with SymRK to regulate downstream cellular responses in Lotus japonicus during the legume-rhizobia symbiosis. Here, we show that SIE3 interacts with SIP1 in both yeast cells and Nicotiana benthamiana. SIE3 associated with itself and formed a homodimer. The cysteine 266 residue was found to be essential for SIE3 dimerization and for promoting nodulation in transgenic hairy roots of L. japonicus. Our findings provide a foundation for further investigating the regulatory mechanisms of the SymRK-mediated signaling pathway, as well as the biological function of E3 ligase dimerization in nodule organogenesis.

Grain shape in rice is a key determinant of grain appearance, yield and market value, and thus has been widely studied. Rice mutant lines with long-grain phenotypes were previously isolated from an M2 population derived from mutagenized mature pollen grains that were treated with gamma-ray irradiation for a cultivar Ma85. To understand the genetic basis underlying the long-grain trait, two mutant lines, JF171 and JF178, were crossed with the short-grain parents JF222 and Samba, generating three F2 populations. Molecular marker-based genetic analysis was employed to detect the major QTLs that affected grain length, grain width, length-to-width ratio and grain weight. Based on the data obtained for the three populations, a joint major QTL for grain length was mapped to a 4.7 Mb region between the SSR makers RM263 and RM318 on the long arm of chromosome 2. The results suggested that the two independent mutations contained in JF171 and JF178 are likely due to alterations in the same genetic region. Furthermore, we developed a BC2F2 population using JF178 and Samba and narrowed the region to 0.6 Mb. The results of the current study will be helpful to reveal the genetic basis of the above long-grain mutant lines. In general, our results provide solid genetic data to identify the unknown gene that affects grain length by map-based cloning and practice marker-assisted breeding for long-grain rice cultivars.

Barley awns are highly active in photosynthesis and account for 30–50% of grain weight in barley. They are diverse in length, ranging from long to awnless, and in shape from straight to hooded or crooked. Their diversity and importance have intrigued geneticists for several decades. A large collection of awnness mutants are available—over a dozen of them have been mapped on chromosomes and a few recently cloned. Different awnness genes interact with each other to produce diverse awn phenotypes. With the availability of the sequenced barley genome and application of new mapping and gene cloning strategies, it will now be possible to identify and clone more awnness genes. A better understanding of the genetic basis of awn diversity will greatly facilitate development of new barley cultivars with improved yield, adaptability and sustainability.

OsWUS has recently been shown to be a transcription factor gene critical for tiller development and fertility in rice. The OsWUS protein consists of three conserved structural domains, but their biological functions are still unclear. We discovered a new rice mutant resulting from tissue culture, which hardly produced tillers and exhibited complete female sterility. The male and female floral organs of the mutant were morphologically indistinguishable from those of the wild type. We named the mutant srt1 for completely sterile and reduced tillering 1. Map-based cloning revealed that the mutant phenotypes were caused by a mutation in OsWUS. Compared with the two previously reported null allelic mutants of OsWUS (tab1-1 and moc3-1), which could produce partial N-terminal peptides of OsWUS, the srt1 protein contained a deletion of only seven amino acids within the conserved homeobox domain of OsWUS. However, the mutant phenotypes (monoculm and female sterility) displayed in srt1 were as typical and severe as those in tab1-1 and moc3-1. This indicates that the homeobox domain of SRT1 is essential for the regulation of tillering and sterility in rice. In addition, srt1 showed an opposite effect on panicle development to that of the two null allelic mutants, implying that the srt1 protein might still have partial or even new functions on panicle development. The results of this study suggest that the homeobox domain is pivotal for OsWUS function.

A mutation in Large Grain Size 1 results in increases in grain length, grain width, grain weight, and endosperm cell number, as well as enhancement of cold tolerance in rice., Grain shape is controlled by quantitative trait loci (QTLs) in rice (Oryza sativa L.). A rice mutant (JF178) with long and large grains has been used in a breeding program for over a decade, but its genetic basis has been unclear. Here, a semi-dominant QTL, designated Large Grain Size 1 (LGS1), was cloned and the potential molecular mechanism of LGS1 function was studied. Near-isogenic lines (NILs) and a map-based approach were employed to clone the LGS1 locus. LGS1 encodes the OsGRF4 transcription factor and contains a 2 bp missense mutation in the coding region that coincides with the putative pairing site of miRNA396. The LGS1 transcript levels in the mutant line were found to be higher than the lgs1 transcript levels in the control plants, suggesting that the mutation might disrupt the pairing of the LGS1 mRNA with miR396. In addition to producing larger grains, LGS1 also enhanced cold tolerance at the seedling stage and increased the survival rate of seedlings after cold stress treatment. These findings indicate that the mutation in LGS1 appears to disturb the GRF4–miR396 stress response network and results in the development of enlarged grains and enhancement of cold tolerance in rice.

Symbiosis receptor-like kinase (SymRK) is a key protein mediating the legume-Rhizobium symbiosis. Our previous work has identified an MAP kinase kinase, SIP2, as a SymRK-interacting protein to positively regulate nodule organogenesis in Lotus japonicus, suggesting that an MAPK cascade might be involved in Rhizobium-legume symbiosis. In this study, LjMPK6 was identified as a phosphorylation target of SIP2. Stable transgenic L. japonicus with RNAi silencing of LjMPK6 decreased the numbers of nodule primordia (NP) and nodule, while plants overexpressing LjMPK6 increased the numbers of nodule, infection threads (ITs), and NP, indicating that LjMPK6 plays a positive role in nodulation. LjMPK6 could interact with a cytokinin receptor, LHK1 both in vivo and in vitro. LjMPK6 was shown to compete with LHP1 to bind to the receiver domain (RD) of LHK1and to downregulate the expression of two LjACS (1-aminocyclopropane-1-carboxylic acid synthase) genes and ethylene levels during nodulation. This study demonstrated an important role of LjMPK6 in regulation of nodule organogenesis and ethylene production in L. japonicus.

Background In mammals, nucleostemin (NS), a nucleolar GTPase, is involved in stem cell proliferation, embryogenesis and ribosome biogenesis. Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) has previously been shown to be essential for plant growth and development. However, the role of NSN1 in cell proliferation is largely unknown. Results Using nsn1, a loss-of-function mutant of Arabidopsis NSN1, we investigated the function of NSN1 in plant cell proliferation and cell cycle regulation. Morphologically, nsn1 exhibited developmental defects in both leaves and roots, producing severely reduced vegetative organs with a much smaller number of cells than those in the wild type. Dynamic analysis of leaf and root growth revealed a lower cell proliferation rate and slower cell division in nsn1. Consistently, the transcriptional levels of key cell cycle genes, including those regulating the transition of G1-S and G2-M, were reduced drastically in nsn1. The introduction of CYCLIN B1::GUS into nsn1 resulted in confined expression of GUS in both the leaf primordia and root meristem, indicating that cell proliferation was hampered by the mutation of NSN1. Upon subjection to treatment with bleomycin and methyl methanesulfonate (MMS), nsn1 plants exhibited hypersensitivity to the genotoxic agents. In the nucleus, NSN1 interacted with nucleosome assembly protein1 (AtNAP1;1), a highly conserved histone chaperone functioning in cell proliferation. Notably, the N-terminal conserved domains of Arabidopsis NSN1 were critical for the physical interaction. Conclusions As a conserved homolog of mammalian nucleostemin, Arabidopsis NSN1 plays pivotal roles in embryogenesis and ribosome biogenesis. In this study, NSN1 was found to function as a positive regulator in cell cycle progression. The interaction between NSN1 and histone chaperone AtNAP1;1, and the high resemblance in sensitivity to genotoxics between nsn1 and atnap1;1 imply the indispensability of the two nuclear proteins for cell cycle regulation. This work provides an insight into the delicate control of cell proliferation through the cooperation of a GTP-binding protein with a nucleosome assembly/disassembly protein in Arabidopsis. Electronic supplementary material The online version of this article (10.1186/s12870-018-1289-2) contains supplementary material, which is available to authorized users.

Table S1. Analysis of protein identity among AtNRP1s and AtNRPs. (PDF 98Â kb)

Phytosulfokine (PSK) is a tyrosine-sulfated peptide that is widely distributed in plants, participating in cell proliferation, differentiation, and innate immunity. The potential role of PSK in nodulation in legumes has not been reported. In this work, five PSK precursor genes were identified in Lotus japonicas, designated as LjPSK1 to LjPSK5. Three of them (LjPSK1, LjPSK4, and LjPSK5) were found to be expressed in nitrogen-fixing root nodules. LjPSK1 and LjPSK4 were not induced at the early stage of nodulation. Interestingly, while the expression of LjPSK4 was also found in spontaneous nodules without rhizobial colonization, LjPSK1 was not induced in these pseudo nodules. Promoter-β-glucuronidase analysis revealed that LjPSK1 was highly expressed in enlarged symbiotic cells of nodules. Exogenous addition of 1 μM synthetic PSK peptide resulted in increased nodule numbers per plant. Consistently, the number of mature nodules but not the events of rhizobial infection and nodule initiation was increased by overexpressing LjPSK1 in transgenic hairy roots, in which the expression of jasmonate-responsive genes was found to be repressed. These results suggest that PSK is a new peptide signal that regulates nodulation in legumes, probably through cross-talking with other phytohormones.

The calcium/calmodulin-dependent protein kinase CCaMK forms a complex with its phosphorylation target CIP73 (CCaMK-interacting protein of 73 kDa). In this work, a homolog of the animal HSC/HSP70 interacting protein (HIP) was identified as an interacting partner of CIP73 in Lotus japonicus. L. japonicus HIP contains all functional domains characteristic of animal HIP proteins. The C-terminal STI1-like domain of L. japonicus HIP was found to be necessary and sufficient for interaction with CIP73. The interaction between CIP73 and HIP occurred in both the nuclei and cytoplasm in Nicotiana benthamiana leaf cells. The interactions between CIP73 and HIP and between CIP73 and CCaMK could take place simultaneously in the same nuclei. HIP transcripts were detected in all plant tissues tested. As nodule primordia developed into young nodules, the expression of HIP was down-regulated and the HIP transcript level became very low in mature nodules. More nodules were formed in transgenic hairy roots of L. japonicus expressing HIP RNA interference at 16 days postinoculation as compared with the control hairy roots expressing the empty vector. It appears that HIP may play a role as a negative regulator for nodulation.

Mechanisms underlying nodulation factor signaling downstream of the nodulation factor receptors (NFRs) have not been fully characterized. In this study, clathrin heavy chain1 (CHC1) was shown to interact with the Rho-Like GTPase ROP6, an interaction partner of NFR5 in Lotus japonicus. The CHC1 gene was found to be expressed constitutively in all plant tissues and induced in Mesorhizobium loti-infected root hairs and nodule primordia. When expressed in leaves of Nicotiana benthamiana, CHC1 and ROP6 were colocalized at the cell circumference and within cytoplasmic punctate structures. In M. loti-infected root hairs, the CHC protein was detected in cytoplasmic punctate structures near the infection pocket along the infection thread membrane and the plasma membrane of the host cells. Transgenic plants expressing the CHC1-Hub domain, a dominant negative effector of clathrin-mediated endocytosis, were found to suppress early nodulation gene expression and impair M. loti infection, resulting in reduced nodulation. Treatment with tyrphostin A23, an inhibitor of clathrin-mediated endocytosis of plasma membrane cargoes, had a similar effect on down-regulation of early nodulation genes. These findings show an important role of clathrin in the leguminous symbiosis with rhizobia.

Barley m38 mutant was selected for its high level of mixed-linkage (1,3),(1,4)-β-d-glucan (MLG) in the grain. This elevated level of MLG was found to be associated with decreased amylose accumulation as well as other chemical composition alterations. Molecular characterization results revealed m38 as a new allele of the Waxy gene, encoding an endosperm-specific granule-bound starch synthase I (GBSSI). Additional mapping data from amylose phenotype and GBSSI gene specific markers supported the conclusion of the GBSSI mutation in m38. The m38 locus contains a nucleotide alteration that would result in the substitution of glycine at position 263 with serine in the putative adenosine-5′-diphosphate-glucose binding domain. This amino acid substitution alters loop structures on the exterior surface of the folded protein and may affect its enzyme activity. Characterizations of m38 in this report provide for a new allele of the Waxy gene and additional evidence of pleiotropic effects on other chemical co...

The chemical induced barley mutant m351 was first selected for its low level of mixed-linkage (1–3,1–4) beta-D-glucan (MLG) in an experimental effort to search for barley lines with varied grain MLG contents. The MLG decrease in m351 was associated with increased levels of fructans and crude fiber, but maintained the same plant characteristics under field conditions. The mutation was mapped to a genetic locus flanked by two SSR markers, Bmag369 and Bmag564, on chromosome 7H. Molecular cloning of the CslF6 gene from the m351 line revealed the presence of a point mutation, causing a substitution of an alanine for threonine at position 849 in the amino acid sequence of the corresponding protein. The resultant protein retains some functionality and affects other components in the m351 grain. Those metabolic changes associated with MLG reduction in m351 is the first case reported of a partially functional CslF6 gene in cereal grains. The results contribute to better understanding of the functional effects of the CslF6 gene and the mutant has potential implications in grain end-use quality improvement.

Summary Transcription factor complex formation is a central step in regulating gene expression. In this report, a novel MYB coiled-coil transcription factor referred to as IPN2, for Interacting Protein of NSP2, is described. The interaction between IPN2 and NSP2 was examined by protein pull-down assays and bimolecular fluorescence complementation (BiFC). Subcellular localization of proteins, gene expression and gene function were assessed in transgenic hairy roots expressing tagged recombinant proteins, promoter-reporter and RNA interference (RNAi) constructs, respectively. The GRAS domain of NSP2 and the coiled-coil domain of IPN2 were found to be responsible for the interaction between the two proteins. IPN2 had strong transcription activation activity, bound directly to the NIN gene promoter, and was localized to the nuclei of Lotus japonicus root cells. The expression of IPN2 was elevated during nodule development, coinciding with increased NSP2 gene expression during nodule organogenesis. RNAi-mediated knockdown expression of IPN2 did not affect arbuscular mycorrhizal development, but had deleterious effects on rhizobial infection and nodule formation in L. japonicus. These results demonstrate an important role of IPN2 in nodule organogenesis and place a new MYB transcription factor in the Nod signaling pathway.

The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus.

The symbiotic interaction between legume plants and rhizobia results in the formation of root nodules, in which symbiotic plant cells host and harbor thousands of nitrogen-fixing rhizobia. Here, a Medicago truncatula nodules with activated defense 1 (nad1) mutant was identified using reverse genetics methods. The mutant phenotype was characterized using cell and molecular biology approaches. An RNA-sequencing technique was used to analyze the transcriptomic reprogramming of nad1 mutant nodules. In the nad1 mutant plants, rhizobial infection and propagation in infection threads are normal, whereas rhizobia and their symbiotic plant cells become necrotic immediately after rhizobia are released from infection threads into symbiotic cells of nodules. Defense-associated responses were detected in nad1 nodules. NAD1 is specifically present in root nodule symbiosis plants with the exception of Morus notabilis, and the transcript is highly induced in nodules. NAD1 encodes a small uncharacterized protein with two predicted transmembrane helices and is localized at the endoplasmic reticulum. Our data demonstrate a positive role for NAD1 in the maintenance of rhizobial endosymbiosis during nodulation.

Nod Factor Receptor5 (NFR5) is an atypical receptor-like kinase, having no activation loop in the protein kinase domain. It forms a heterodimer with NFR1 and is required for the early plant responses to Rhizobium infection. A Rho-like small GTPase from Lotus japonicus was identified as an NFR5-interacting protein. The amino acid sequence of this Rho-like GTPase is closest to the Arabidopsis (Arabidopsis thaliana) ROP6 and Medicago truncatula ROP6 and was designated as LjROP6. The interaction between Rop6 and NFR5 occurred both in vitro and in planta. No interaction between Rop6 and NFR1 was observed. Green fluorescent protein-tagged ROP6 was localized at the plasma membrane and cytoplasm. The interaction between ROP6 and NFR5 appeared to take place at the plasma membrane. The expression of the ROP6 gene could be detected in vascular tissues of Lotus roots. After inoculation with Mesorhizobium loti, elevated levels of ROP6 expression were found in the root hairs, root tips, vascular bundles of roots, nodule primordia, and young nodules. In transgenic hairy roots expressing ROP6 RNA interference constructs, Rhizobium entry into the root hairs did not appear to be affected, but infection thread growth through the root cortex were severely inhibited, resulting in the development of fewer nodules per plant. These data demonstrate a role of ROP6 as a positive regulator of infection thread formation and nodulation in L. japonicus.

Callose is the major polysaccharide present in the callose wall of developing microspores and the growing pollen tube wall. It is also an essential component of other specialized cell walls and its synthesis can be induced by pathogen infection, wounding and environmental cues. Among the 12 callose synthase genes (CalS) present in the Arabidopsis genome, CalS5 plays the predominant role in the synthesis of the callose wall, callose plugs and pollen tube wall. When expressed as a GFP-tagged protein in cultured tobacco BY-2 cells, CalS5 was found to be present in the plasma membrane and the Golgi-related endomembranes. Unlike the cell plate-specific CalS1 isozyme, CalS5 was not concentrated to the cell plate at cytokinesis. Expression of CalS5 resulted in callose accumulation only in the cell wall of BY-2 cells. The fact that no callose was found in the endomembranes suggests that CalS5 is not functional in that compartment. These cells exhibited a decreased plasmolysis rate in hypotonic solutions and an increased cytolysis rate in hypertonic conditions. This study demonstrates that an artificial callose wall could be synthesized by expressing a callose synthase enzyme.

Arabidopsis NSN1 encodes a nucleolar GTP-binding protein and is required for flower development. Defective flowers were formed in heterozygous nsn1/+ plants. Homozygous nsn1 plants were dwarf and exhibited severe defects in reproduction. Arrests in embryo development in nsn1 could occur at any stage of embryogenesis. Cotyledon initiation and development during embryogenesis were distorted in nsn1 plants. At the seedling stage, cotyledons and leaves of nsn1 formed upward curls. The curled leaves developed meristem-like outgrowths or hyperplasia tissues in the adaxial epidermis. Long and enlarged pavement cells, characteristic of the abaxial epidermis of wild type plants, were found in the adaxial epidermis in nsn1 leaves, suggesting a disoriented leaf polarity in the mutant. The important role of NSN1 in embryo development and leaf differentiation was consistent with the high level expression of the NSN1 gene in the developing embryos and the primordia of cotyledons and leaves. The CLAVATA 3 (CLV3) gene, a stem cell marker in the Arabidopsis shoot apical meristem (SAM), was expressed in expanded regions surrounding the SAM of nsn1 plants, and induced ectopically in the meristem-like outgrowths in cotyledons and leaves. The nsn1 mutation up-regulated the expression levels of several genes implicated in the meristem identity and the abaxial cell fate, and repressed the expression of other genes related to the specification of cotyledon boundary and abaxial identity. These results demonstrate that NSN1 represents a novel GTPase required for embryogenesis, leaf development and leaf polarity establishment in Arabidopsis.

In the Rhizobium-legume symbiosis, calcium/calmodulin-dependent protein kinase (CCaMK) is a key regulator for both rhizobial infection and nodule organogenesis. Deregulation of CCaMK by either a point mutation in the autophosphorylation site or the deletion of the carboxyl-terminal regulatory domain results in spontaneous nodule formation without rhizobia. However, the underlying biochemical mechanisms are poorly understood. Here, using the kinase domain of CCaMK as a bait in yeast two-hybrid screening, we identify a novel protein, CIP73 (for CCaMK-interacting protein of approximately 73 kD), that interacts with CCaMK. CIP73 contains a Scythe_N ubiquitin-like domain and belongs to the large ubiquitin superfamily. Deletion and mutagenesis analysis demonstrate that CIP73 could only interact with CCaMK when the calmodulin-binding domain and three EF-hand motifs are removed from the kinase domain. The amino-terminal 80 amino acid residues (80–160) of CCaMK are required for interacting with CIP73 in yeast cells. On the other hand, protein pull-down assay and bimolecular fluorescence complementation assay in Nicotiana benthamiana show that the full-length CCaMK could interact with CIP73 in vitro and in planta. Importantly, CCaMK phosphorylates the amino terminus of CIP73 in a Ca2+/calmodulin-dependent manner in vitro. CIP73 transcripts are preferentially expressed in roots, and very low expression is detected in leaves, stems, and nodules. The expression in roots is significantly decreased after inoculation of Mesorhizobium loti. RNA interference knockdown of CIP73 expression by hairy root transformation in Lotus japonicus led to decreased nodule formation, suggesting that CIP73 performed an essential role in nodulation.

It has been known for more than a century that sieve plates in the phloem in plants contain callose, a β-1,3-glucan. However, the genes responsible for callose deposition in this subcellular location have not been identified. In this paper we examine callose deposition patterns in T-DNA insertion mutants (cs7) of the Callose Synthase 7 (CalS7) gene. We demonstrated here that the CalS7 gene is expressed specifically in the phloem of vascular tissues. Callose deposition in the phloem, especially in the sieve elements, was greatly reduced in cs7 mutants. Ultrastructural analysis of developing sieve elements revealed that callose failed to accumulate in the plasmodesmata of incipient sieve plates at the early perforation stage of phloem development, resulting in the formation of sieve plates with fewer pores. In wild-type Arabidopsis plants, callose is present as a constituent polysaccharide in the phloem of the stem, and its accumulation can also be induced by wounding. Callose accumulation in both conditions was eliminated in mature sieve plates of cs7 mutants. These results demonstrate that CalS7 is a phloem-specific callose synthase gene, and is responsible for callose deposition in developing sieve elements during phloem formation and in mature phloem induced by wounding. The mutant plants exhibited moderate reduction in seedling height and produced aberrant pollen grains and short siliques with aborted embryos, suggesting that CalS7 also plays a role in plant growth and reproduction.

During the establishment of symbiosis in legume roots, the rhizobial Nod factor signal is perceived by the host cells via receptor-like kinases, including SymRK. The NODULE INCEPTION (NIN) gene in Lotus japonicus is required for rhizobial entry into root cells and for nodule organogenesis. We describe here a novel DNA-binding protein from L. japonicus, referred to as SIP1, because it was identified as a SymRK-interacting protein. SIP1 contains a conserved AT-rich interaction domain (ARID) and represents a unique member of the ARID-containing proteins in plants. The C terminus of SIP1 was found to be responsible for its interaction with the kinase domain of SymRK and for homodimerization in the absence of DNA. SIP1 specifically binds to the promoter of LjNIN but not to that of LjCBP1 (a calcium-binding protein gene), both of which are known to be inducible by Nod factors. SIP1 recognizes two of the three AT-rich domains present in the NIN gene promoter. Deletion of one of the AT-rich domains at the NIN promoter diminishes the binding of SIP1 to the NIN promoter. The protein is localized to the nuclei when expressed as a red fluorescence fusion protein in the onion (Allium cepa) epidermal cells. The SIP1 gene is expressed constitutively in the uninfected roots, and its expression levels are elevated after infection by Mesorhizobium loti. It is proposed that SIP1 may be required for the expression of NIN and involved in the initial communications between the rhizobia and the host root cells.

Using phragmoplastin-interacting protein 1 (PhrIP1) as bait, we isolated an Arabidopsis cDNA encoding Ran2, a small Ras-like GTP-binding protein. The interaction between PhrIP1 and Ran2 was confirmed by an in vitro protein–protein interaction assay with purified Ran2 and PhrIP1. The plant Ran2 shares high sequence homology, 78 and 86% at the amino acid level, with human Ran/TC4 and C. elegans Ran, respectively. Our results obtained from enzyme assays and Western blot analysis show that Ran2 has intrinsic GTPase activity and is present in the soluble fraction of Arabidopsis seedling extract. Fluorescent microscopy using anti-Ran2 antibody revealed that the Ran protein is localized in the perinuclear region with the highest concentration at the nuclear envelope. In contrast to its animal counterparts that are present in the nucleoplasm, the Ran protein is absent inside the nucleus. These results suggest that plant Ran proteins may be involved in mediation of nucleocytoplasmic transport and assembly of the nuclear envelope after karyokinesis in plant cells.

The symbiotic association between the legume Lotus japonicus and the nitrogen-fixing bacterium Mesorhizobium loti results in the formation of root nodules. This process begins with the recognition of the rhizobial nodulation factor (NF) by the NF receptors (NFR) at the cell surface of the host roots. The downstream signaling cascades after NFR recognition have not been fully characterized. We recently identified a clathrin heavy chain 1 (CHC1) from L. japonicus as a potential target of the NF signaling cascades. CHC is a known central component in the clathrin-mediated endocytosis (CME) in eukaryotic cells. The CHC1 gene was highly expressed in Rhizobium-infected root hairs and the CHC1 protein was present in cytoplasmic punctate structures near the infection pockets and along the infection thread membrane. Furthermore, expression of a dominant-negative variant of CHC1 or treatment with a chemical inhibitor of CME resulted in impaired phenotypes in the NF signaling, rhizobial infection and nodulation. These findings open a new avenue for future work aiming at understanding the role of endocytosis in NF signaling pathway and rhizobial infection.

Summary Callose (β-1,3-glucan) is produced at different locations in response to biotic and abiotic cues. Arabidopsis contains 12 genes encoding callose synthase (CalS). We demonstrate that one of these genes, CalS5, encodes a callose synthase which is responsible for the synthesis of callose deposited at the primary cell wall of meiocytes, tetrads and microspores, and the expression of this gene is essential for exine formation in pollen wall. CalS5 encodes a transmembrane protein of 1923 amino acid residues with a molecular mass of 220 kDa. Knockout mutations of the CalS5 gene by T-DNA insertion resulted in a severe reduction in fertility. The reduced fertility in the cals5 mutants is attributed to the degeneration of microspores. However, megagametogenesis is not affected and the female gametes are completely fertile in cals5 mutants. The CalS5 gene is also expressed in other organs with the highest expression in meiocytes, tetrads, microspores and mature pollen. Callose deposition in the cals5 mutant was nearly completely lacking, suggesting that this gene is essential for the synthesis of callose in these tissues. As a result, the pollen exine wall was not formed properly, affecting the baculae and tectum structure and tryphine was deposited randomly as globular structures. These data suggest that callose synthesis has a vital function in building a properly sculpted exine, the integrity of which is essential for pollen viability.

We have characterized 4 of the 16 members of the family of dynamin-related proteins (DRP) in Arabidopsis. Three members, DRP1A (previously referred as ADL1), DRP1C and DRP1E, belong to the largest group of phragmoplastin-like proteins. DRP2A (ADL6) is one of the two members that contain a pleckstrin homology (PH) domain and a proline-rich (PR) motif, characteristics of animal dynamins. All four proteins interacted in yeast two-hybrid assays with phragmoplastin, and showed different patterns of localization at the forming cell plate during cytokinesis. GFP-tagged DRP1A and DRP1C proteins were found to be associated with the cytoskeleton in G1 phase of the cell cycle. The distribution pattern of DRP1A was sensitive to propyzamid and insensitive to cytochalasin D, suggesting that DRP1A is associated with microtubules and not actin filaments. The association of DRP1A with microtubules was confirmed in vitro by spin-down assays. A GTPase-defective phragmoplastin acted as a dominant negative mutant, reduced transport of vesicles to the cell plate and formed dense tubule-like structures in the cell plate. We propose that DRP1 proteins may provide an anchor for Golgi-derived vesicles to attach to microtubules, which in turn direct the vesicles to the forming cell plate during cytokinesis. Whereas the DRP1 subfamily members are involved in tubulization of membranes, DRP2 may be involved in endocytosis and membrane recycling via clathrin-coated vesicles.

Phragmoplastin, a dynamin-like GTPase, is associated with cell plate formation in plants. We expressed a phragmoplastin-GFP construct (Phr G ) under the control of a 35S promoter in transgenic tobacco plants. High levels of expression of this chimeric protein in transgenic plants were confirmed by Western blot analysis. T1 seedlings formed morphologically normal cotyledons, but most were unable to develop beyond the first pair of true leaves. Only 5–10% of the seedlings could develop into small plants whose progenies continued to exhibit severe growth defects. The primary root growth was arrested after 7 days and no lateral roots were formed. The mature primary root meristem consisted of disorganized isodiametrically enlarged cells and no recognizable quiescent center or collumela. Adventitious root primordia were initiated in the hypocotyl, but completely arrested at an early stage and contained differentiated treachery elements. The orientation of cell plates was abnormal, suggesting a possible defect in its positioning. However, unlike other cytokinesis mutants, no binucleate or polyploid cells were found. Fluorescence image cytometry studies indicated that Phr G root meristem cells had 2C levels of DNA and appear to be arrested in G1 stage of the cell cycle. Heavy callose deposition was observed at the nascent cell plate and callose accumulation persisted in new cell walls. Electron microscopy revealed unusual electron dense substances in the maturing cell plate and accumulation of multivesicular bodies around the cell plate. Since phragmoplastin interacts with callose synthase complex, its overexpression may affect accumulation of callose arresting plant growth due to perturbation in cell division progression.

The presence of callose in sieve plates has been known for a long time, but how this polysaccharide plug is synthesized has remained unsolved. Two independent laboratories have recently reported the identification of callose synthase 7 (CalS7), also known as glucan synthase-like 7 (GSL7), as the enzyme responsible for callose deposition in sieve plates. Mutant plants defective in this enzyme failed to synthesize callose in developing sieve plates during phloem formation and were unable to accumulate callose in sieve pores in response to stress treatments. The mutant plants developed less open pores per sieve plate and the pores were smaller in diameter. As a result, phloem conductivity was reduced significantly and the mutant plants were shorter and set fewer seeds.

Callose is synthesized on the forming cell plate and several other locations in the plant. We cloned an Arabidopsis cDNA encoding a callose synthase (CalS1) catalytic subunit. The CalS1 gene comprises 42 exons with 41 introns and is transcribed into a 6.0-kb mRNA. The deduced peptide, with an approximate molecular mass of 226 kD, showed sequence homology with the yeast 1,3-β-glucan synthases and is distinct from plant cellulose synthases. CalS1 contains 16 predicted transmembrane helices with the N-terminal region and a large central loop facing the cytoplasm. CalS1 interacts with two cell plate–associated proteins, phragmoplastin and a novel UDP-glucose transferase that copurifies with the CalS complex. That CalS1 is a cell plate–specific enzyme is demonstrated by the observations that the green fluorescent protein–CalS1 fusion protein was localized at the growing cell plate, that expression of CalS1 in transgenic tobacco cells enhanced callose synthesis on the forming cell plate, and that these cell lines exhibited higher levels of CalS activity. These data also suggest that plant CalS may form a complex with UDP-glucose transferase to facilitate the transfer of substrate for callose synthesis.