Zonglie Hong

A Cell Plate–Specific Callose Synthase and Its Interaction with Phragmoplastin

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.

A Novel UDP-Glucose Transferase Is Part of the Callose Synthase Complex and Interacts with Phragmoplastin at the Forming Cell Plate

Using phragmoplastin as a bait, we isolated an Arabidopsis cDNA encoding a novel UDP-glucose transferase (UGT1). This interaction was confirmed by an in vitro protein–protein interaction assay using purified UGT1 and radiolabeled phragmoplastin. Protein gel blot results revealed that UGT1 is associated with the membrane fraction and copurified with the product-entrapped callose synthase complex. These data suggest that UGT1 may act as a subunit of callose synthase that uses UDP-glucose to synthesize callose, a 1,3-β-glucan. UGT1 also interacted with Rop1, a Rho-like protein, and this interaction occurred only in its GTP-bound configuration, suggesting that the plant callose synthase may be regulated by Rop1 through the interaction with UGT1. The green fluorescent protein–UGT1 fusion protein was located on the forming cell plate during cytokinesis. We propose that UGT1 may transfer UDP-glucose from sucrose synthase to the callose synthase and thus help form a substrate channel for the synthesis of callose at the forming cell plate.

Dysregulation of cell-to-cell connectivity and stomatal patterning by loss-of-function mutation in Arabidopsis CHORUS (GLUCAN SYNTHASE-LIKE 8)

Patterning of stomata, valves on the plant epidermis, requires the orchestrated actions of signaling components and cell-fate determinants. To understand the regulation of stomatal patterning, we performed a genetic screen using a background that partially lacks stomatal signaling receptors. Here, we report the isolation and characterization of chorus (chor), which confers excessive proliferation of stomatal-lineage cells mediated by SPEECHLESS (SPCH). chor breaks redundancy among three ERECTA family genes and strongly enhances stomatal patterning defects caused by loss-of-function in TOO MANY MOUTHS. chor seedlings also exhibit incomplete cytokinesis and growth defects, including disruptions in root tissue patterning and root hair cell morphogenesis. CHOR encodes a putative callose synthase, GLUCAN SYNTHASE-LIKE 8 (GSL8), that is required for callose deposition at the cell plate, cell wall and plasmodesmata. Consistently, symplastic macromolecular diffusion between epidermal cells is significantly increased in chor, and proteins that do not normally move cell-to-cell, including a fluorescent protein-tagged SPCH, diffuse to neighboring cells. Such a phenotype is not a general trait caused by cytokinesis defects. Our findings suggest that the restriction of symplastic movement might be an essential step for the proper segregation of cell-fate determinants during stomatal development.

Genetic bases of rice grain shape: so many genes, so little known

Rice (Oryza sativa) grain shape is a key determinant of grain yield and market values. Facilitated by advancements in genomics and various molecular markers, more than 400 quantitative trait loci (QTLs) associated with rice grain traits have been identified. In this review, we examine the genetic bases of rice grain shape, focusing on the protein products of 13 genes that have been cloned and the chromosome locations of 15 QTLs that have been fine mapped. Although more genes affecting grain traits are likely to be cloned in the near future, characterizing their functions at the biochemical level and applying these molecular data to rice breeding programs will be a more challenging task.

A unified nomenclature for Arabidopsis dynamin-related large GTPases based on homology and possible functions

Z. Hong1, S.Y. Bednarek2, E. Blumwald3, I. Hwang4, G. Jurgens5, D. Menzel6, K.W. Osteryoung7, N.V. Raikhel8, K. Shinozaki9, N. Tsutsumi10 and D.P.S. Verma1,∗ 1Department of Molecular Genetics and Plant Biotechnology Center, Ohio State University, Columbus, OH 43210, USA (∗author for correspondence; e-mail verma.1@osu.edu; 2Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA; 3Department of Pomology, University of California, One Shields Ave., Davis, CA 95616, USA; 4Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, 790-784, Korea; 5Lehrstuhl fur Entwicklungsgenetik, Universitat Tubingen, 72076 Tubingen, Germany; 6Zellbiologie der Pflanzen, Botanisches Institut, Universitat Bonn, Kirschallee 1, Bonn, 53115, Germany; 7Department of Plant Biology, 166 Plant Biology Building, Michigan State University, East Lansing, MI 48824, USA; 8Department of Botany and Plant Sciences and Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA; 9Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba 305-0074, Japan; 10Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

CalS7 encodes a callose synthase responsible for callose deposition in the phloem

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.

Expression of callose synthase genes and its connection with Npr1 signaling pathway during pathogen infection

Callose synthesis occurs at specific stages of plant cell wall development in all cell types, and in response to pathogen attack, wounding and physiological stresses. We determined the expression pattern of “upstream regulatory sequence” of 12 Arabidopsis callose synthase genes (CalS1–12) genes and demonstrated that different callose synthases are expressed specifically in different tissues during plant development. That multiple CalS genes are expressed in the same cell type suggests the possibility that CalS complex may be constituted by heteromeric subunits. Five CalS genes were induced by pathogen (Hyaloperonospora arabidopsis, previously known as Peronospora parasitica, the causal agent of downy mildew) or salicylic acid (SA), while the other seven CalS genes were not affected by these treatments. Among the genes that are induced, CalS1 and CalS12 showed the highest responses. In Arabidopsis npr1 mutant, impaired in response of pathogenesis related (PR) genes to SA, the induction of CalS1 and CalS12 genes by the SA or pathogen treatments was significantly reduced. The patterns of expression of the other three CalS genes were not changed significantly in the npr1 mutant. These results suggest that the high induction observed of CalS1 and CalS12 is Npr1 dependent while the weak induction of five CalS genes is Npr1 independent. In a T-DNA knockout mutant of CalS12, callose encasement around the haustoria on the infected leaves was reduced and the mutant was found to be more resistant to downy mildew as compared to the wild type plants.

Biogenesis of the peribacteriod membrane in root nodules

An infected root nodule cell may contain several thousand rhizobial symbionts, each enclosed in a membrane envelope, the peribacteroid membrane (PBM). The PBM is derived from the host plasma membrane, but shares properties with the vacuolar membrane and contains several nodule-specific proteins (nodulins) that perform unique functions for symbiosis.

A Novel ARID DNA-Binding Protein Interacts with SymRK and Is Expressed during Early Nodule Development in Lotus japonicus

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.

Small GTP-Binding Proteins and Membrane Biogenesis in Plants.

One of the amazing features of the cellular machinery is that all organeller and membrane proteins, as well as those destined for secretion, have an attached address label for targeting to a specific site. Following synthesis, these proteins are folded, shipped, delivered, and received at the right compartment. Their assigned functions are performed only when they are properly placed at a designated site in the cell. Membrane vesicles play an essential role in protein transport as carriers of specific proteins to intracellular compartments. This process begins immediately after perception of specific signals and involves membrane ruffling, budding and transport of ER vesicles, fusion and passage through the Golgi, and release of vesicles from trans-Golgi cisternae to target to the vacuoles and plasma membrane. The components of the vesicle-mediated protein trafficking system are not, however, well defined. It is not known what kind of biochemical principles are operative for unidirectional transport of vesicles. How are vesicles fused to the target compartment? Although much remains to be understood, many studies from yeast and mammalian systems have identified some key players in this pathway. Isolation of plant homologs of some of these proteins has confirmed that these steps are conserved in evolution and must involve well-defined reactions. We focus here on the relevance of small GTP-binding proteins in vesicle-mediated protein transport (for earlier reviews, see Balch, 1990; Bednarek and Raikhel, 1992; Pryer et al., 1992; Terryn et al., 1993; Zerial and Stenmark, 1993).