SummaryPre-harvest sprouting (PHS) or vivipary in cereals is an important agronomic trait that results in significant economic loss. A considerable number of mutations that cause PHS have been identified in several species. However, relatively few viviparous mutants in rice (Oryza sativa L.) have been reported. To explore the mechanism of PHS in rice, we carried out an extensive genetic screening and identified 12 PHS mutants (phs). Based on their phenotypes, these phs mutants were classified into three groups. Here we characterize in detail one of these groups, which contains mutations in genes encoding major enzymes of the carotenoid biosynthesis pathway, including phytoene desaturase (OsPDS), f-carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene b-cyclase (b-OsLCY), which are essential for the biosynthesis of carotenoid precursors of ABA. As expected, the amount of ABA was reduced in all four phs mutants compared with that in the wild type. Chlorophyll fluorescence analysis revealed the occurrence of photoinhibition in the photosystem and decreased capacity for eliminating excess energy by thermal dissipation. The greatly increased activities of reactive oxygen species (ROS) scavenging enzymes, and reduced photosystem (PS) II core proteins CP43, CP47 and D1 in leaves of the Oscrtiso/phs3-1mutant and OsLCY RNAi transgenic rice indicated that photo-oxidative damage occurred in PS II, consistent with the accumulation of ROS in these plants. These results suggest that the impairment of carotenoid biosynthesis causes photo-oxidation and ABA-deficiency phenotypes, of which the latter is a major factor controlling the PHS trait in rice.

MicroRNA319 (miR319) family is one of the conserved microRNA (miRNA) families among diverse plant species. It has been reported that miR319 regulates plant development in dicotyledons, but little is known at present about its functions in monocotyledons. In rice (Oryza sativa L.), the MIR319 gene family comprises two members, Osa-MIR319a and Osa-MIR319b. Here, we report an expression pattern analysis and a functional characterization of the two Osa-MIR319 genes in rice. We found that overexpressing OsaMIR319a and Osa-MIR319b in rice both resulted in wider leaf blades. Leaves of osa-miR319 overexpression transgenic plants showed an increased number of longitudinal small veins, which probably accounted for the increased leaf blade width. In addition, we observed that overexpressing osamiR319 led to enhanced cold tolerance (4°C) after chilling acclimation (12°C) in transgenic rice seedlings. Notably, under both 4 and 12°C low temperatures, Osa-MIR319a and Osa-MIR319b were down-regulated while the expression of miR319-targeted genes was induced. Furthermore, genetically down-regulating the expression of either of the two miR319-targeted genes, OsPCF5 and OsPCF8, in RNA interference (RNAi) plants also resulted in enhanced cold tolerance after chilling acclimation. Our findings in this study demonstrate that miR319 plays important roles in leaf morphogenesis and cold tolerance in rice.

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. The two major subspecies of Asian cultivated rice (Oryza sativa L.), indica and japonica, have shown obvious differences in rice blast resistance, but the genomic basis that underlies the difference is not clear. We performed a genomewide comparison of the major class of resistant gene family, the nucleotide-binding site-leucinerich repeat (NBS-LRR) gene family, between 93-11 (indica) and Nipponbare ( japonica) with a focus on their pseudogene members. We found great differences in either constitution or distribution of pseudogenes between the two genomes. According to this comparison, we designed the PCR-based molecular markers specific to the Nipponbare NBS-LRR pseudogene alleles and used them as cosegregation markers for blast susceptibility in a segregation population from a cross between a rice blast-resistant indica variety and a susceptible japonica variety. Through this approach, we identified a new blast resistance gene, Pid3, in the indica variety, Digu. The allelic Pid3 loci in most of the tested japonica varieties were identified as pseudogenes due to a nonsense mutation at the nucleotide position 2208 starting from the translation initiation site. However, this mutation was not found in any of the tested indica varieties, African cultivated rice varieties, or AA genome-containing wild rice species. These results suggest that the pseudogenization of Pid3 in japonica occurred after the divergence of indica and japonica.

SummaryThe two-component system (TCS) composed of a pair of a sensor histidine kinase and a response regulator, allows bacteria to sense signals and respond to changes in their environment through specific gene activation or repression. The present study examined TCS in the obligatory intracellular bacteria Ehrlichia chaffeensis and Anaplasma phagocytophilum , that cause human monocytic ehrlichiosis (HME) and human granulocytic anaplasmosis (HGA) respectively. The genomes of E. chaffeensis and A. phagocytophilum were each predicted to encode three pairs of TCSs. All six genes encoding three histidine kinases and three response regulators were expressed in both E. chaffeensis and A. phagocytophilum cultured in human leukocytes. Pretreatment of host cell-free E. chaffeensis or A. phagocytophilum with closantel, an inhibitor of histidine kinases, completely blocked the infection of host cells. Treatment of infected cells 1 day post infection with closantel cleared infection in dose-dependent manner. All six genes in E. chaffeensis were cloned, recombinant proteins were expressed, and polyclonal antibodies were produced. Double immunofluorescence labelling and Western blot analysis revealed that all six proteins were expressed in cell culture. Autokinase activities of the three recombinant histidine kinases from E. chaffeensis were inhibited by closantel in vitro . A number of E. chaffeensis genes, including the six TCS genes, were downregulated within 5-60 min post closantel treatment. These results suggest that these TCSs play an essential role in infection and survival of E. chaffeensis and A. phagocytophilum in human leukocytes.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

Summary Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Ehrlichiae have a biphasic developmental cycle consisting of dense-cored cells (DCs) and reticulate cells (RCs). Isolated DCs are more stress resistant and infectious than RCs. Here, we report that a response regulator, CtrA was upregulated in human monocytes at the late growth stage when DCs develop. E. chaffeensis CtrA bound to the promoters of late-stage transcribed genes: ctrA, ompA (peptidoglycan-associated lipoprotein), bolA (stress-induced morphogen), and surE (stationary phase survival protein), which contain CtrA-binding motifs, and transactivated ompA, surE, and bolA promoter-lacZ fusions in Escherichia coli. OmpA was predominantly expressed in DCs. E. chaffeensis binding to and subsequent infection of monocytes were inhibited by anti-OmpA IgG. E. chaffeensis BolA bound to the promoters of genes encoding outer surface proteins TRP120 and ECH_1038, which were expressed in DCs, and transactivated trp120 and ECH_1038 promoter-lacZ fusions. E. chaffeensis bolA complemented a stress-sensitive E. coli bolA mutant. E. coli expressing E. chaffeensis surE exhibited increased resistance to osmotic stress. Our results suggest that E. chaffeensis CtrA plays a role in coordinating development of the stress resistance for passage from the present to the next host cells through its regulon.

Polyamines are implicated in regulating various developmental processes in plants, but their exact roles and how they govern these processes still remain elusive. We report here an Arabidopsis bushy and dwarf mutant, bud2, which results from the complete deletion of one member of the small gene family that encodes S-adenosylmethionine decarboxylases (SAMDCs) necessary for the formation of the indispensable intermediate in the polyamine biosynthetic pathway. The bud2 plant has enlarged vascular systems in inflorescences, roots, and petioles, and an altered homeostasis of polyamines. The double mutant of bud2 and samdc1, a knockdown mutant of another SAMDC member, is embryo lethal, demonstrating that SAMDCs are essential for plant embryogenesis. Our results suggest that polyamines are required for the normal growth and development of higher plants.Cell Research (2006) IntroductionPolyamines, including diamine putrescine, triamine spermidine and tetraamine spermine, are aliphatic nitrogen compounds distributed widely from bacteria to higher plants [1,2] and have been implicated to play important roles in growth and development [3]. At the cellular level, polyamines are organic cations, interacting with the macromolecules that possess anionic groups such as DNA, RNA, lipids and proteins, thereby influencing DNA conformation, gene expression and protein synthesis, and modulating enzyme activity. In higher plants, polyamines are able to affect membrane fluidity by binding to phospholipids in membrane [4] and mediate biotic and abiotic stress responses, such as pathogen infection, osmotic stress, potassium deficiency and wounding [5][6][7][8][9]. Previous observations revealed that polyamines may be involved in a variety of plant developmental processes, such as cell division, root initiation, somatic embryogenesis, xylogenesis, flower development, fruit ripening and senescence [3]. Recent studies have indicated that polyamines also affect the formation of plant architecture, such as internode elongation [10,11], root branching [12] and shoot apical dominance [13].The plant polyamine biosynthetic pathway is relatively simple [14]. Putrescine is derived either from ornithine catalyzed by ornithine decarboxylase (ODC) or from arginine through several steps catalyzed by arginine decarboxylase (ADC), agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase. Spermidine and spermine are synthesized from putrescine through spermidine and spermine synthases (SPDS and SPMS) from the donor of decarboxylated S-adenosylmethionine (dcSAM), which is produced from S-adenosylmethionine (SAM) by the action of S-adenosylmethionine decarboxylase (SAMDC).Although application of inhibitors is very useful in studying the polyamine biosynthetic pathway and in ex- [11,[19][20][21][22][23][24]. However, no mutant defective in SAMDC has been reported yet, although SAMDC cDNAs were cloned and their transgenic plants have been generated [25][26][27][28][29]. In this paper, we report the isolation and characterization of an Arabidopsis...

Summary Panicle architecture is one of the most important agronomical traits that directly contribute to grain yield in rice (Oryza sativa L.). We report herein an in‐depth characterization of two allelic larger panicle (lp) mutants that show significantly increased panicle size as well as improved plant architecture. Morphological analyses reveal that panicles of two mutants produced more inflorescence branches, especially the primary branches, and contained more grains. Moreover, mutant plants also display more lodging resistance than the wild type. The grain yield per plant in mutants is also increased, suggesting that mutant plants have useful potential for high grain yield in rice breeding. Map‐based cloning reveals that LARGER PANICLE (LP) encodes a Kelch repeat‐containing F‐box protein. RNA in situ hybridization studies display that LP expression was enriched in the branch primordial region. Subcellular localization analyses demonstrate that LP is an endoplasmic reticulum (ER) localized protein, suggesting that LP might be involved in ER‐associated protein degradation (ERAD). Using yeast two‐hybrid assay and bimolecular fluorescence complementation analysis, we confirm that LP is an F‐box protein and could interact with rice SKP1‐like protein in an F‐box domain‐dependent manner. Quantitative real‐time PCR results show that OsCKX2, which encodes cytokinin oxidase/dehydrogenase, is down‐regulated evidently in mutants, implying that LP might be involved in modulating cytokinin level in plant tissues. These results suggest that LP plays an important role in regulating plant architecture, particularly in regulating panicle architecture, thereby representing promising targets for genetic improvement of grain production plants.