Unconventional origin and hybrid system for construction of pyrrolopyrrole moiety in kosinostatin biosynthesis.

Kosinostatin (KST), an antitumor antibiotic, features a pyrrolopyrrole moiety spirally jointed to a five-membered ring of an anthraquinone framework glycosylated with a γ-branched octose. By a combination of in silico analysis, genetic characterization, biochemical assay, and precursor feeding experiments, a biosynthetic pathway for KST was proposed, which revealed (1) the pyrrolopyrrole moiety originates from nicotinic acid and ribose, (2) the bicyclic amidine is constructed by a process similar to the tryptophan biosynthetic pathway, and (3) a discrete adenylation enzyme and a peptidyl carrier protein (PCP) are responsible for producing a PCP-tethered building block parallel to type II polyketide synthase (PKS) rather than for the PKS priming step by providing the starter unit. These findings provide an opportunity to further explore the inexplicable enzymatic logic that governs the formation of pyrrolopyrrole moiety and the spirocyclic skeleton.

CYCLIN-DEPENDENT KINASE G1 is associated with the spliceosome to regulate CALLOSE SYNTHASE5 splicing and pollen wall formation in Arabidopsis.

Arabidopsis thaliana CYCLIN-DEPEDENT KINASE G1 (CDKG1) belongs to the family of cyclin-dependent protein kinases that were originally characterized as cell cycle regulators in eukaryotes. Here, we report that CDKG1 regulates pre-mRNA splicing of CALLOSE SYNTHASE5 (CalS5) and, therefore, pollen wall formation. The knockout mutant cdkg1 exhibits reduced male fertility with impaired callose synthesis and abnormal pollen wall formation. The sixth intron in CalS5 pre-mRNA, a rare type of intron with a GC 5' splice site, is abnormally spliced in cdkg1. RNA immunoprecipitation analysis suggests that CDKG1 is associated with this intron. CDKG1 contains N-terminal Ser/Arg (RS) motifs and interacts with splicing factor Arginine/Serine-Rich Zinc Knuckle-Containing Protein33 (RSZ33) through its RS region to regulate proper splicing. CDKG1 and RS-containing Zinc Finger Protein22 (SRZ22), a splicing factor interacting with RSZ33 and U1 small nuclear ribonucleoprotein particle (snRNP) component U1-70k, colocalize in nuclear speckles and reside in the same complex. We propose that CDKG1 is recruited to U1 snRNP through RSZ33 to facilitate the splicing of the sixth intron of CalS5.

Identification of phoslactomycin biosynthetic gene clusters from Streptomyces platensis SAM-0654 and characterization of PnR1 and PnR2 as positive transcriptional regulators.

Phoslactomycins (PLMs) are inhibitors of protein serine/threonine phosphatase 2A showing diverse and important antifungal, antibacterial and antitumor activity. PLMs are polyketide natural products and produced by several Streptomyces species. The PLMs biosynthetic gene clusters were identified from Streptomyces platensis SAM-0654 and localized in two separate genomic regions, consisting of 27 open reading frames that encode polyketide synthases (PKSs), enzymes for cyclohexanecarboxyl-CoA (CHC-CoA) and ethylmalonyl-CoA (Em-CoA) synthesis, enzymes for post-PKS modifications, proposed regulators, and putative resistance transporters. Bioinformatic analysis and inactivation experiment of regulatory genes suggest that PnR1 and PnR2 are two positive regulators of PLMs biosynthesis. Gene transcription analysis by reverse transcriptase PCR (RT-PCR) of the PLMs gene cluster demonstrated that PnR1 and PnR2 activate the transcription of the structural biosynthetic genes while PnR2 specially governs the transcription of pnR1 in a higher level.

MULTIPOLAR SPINDLE 1 (MPS1), a novel coiled-coil protein of Arabidopsis thaliana, is required for meiotic spindle organization.

The spindle is essential for chromosome segregation during meiosis, but the molecular mechanism of meiotic spindle organization in higher plants is still not well understood. Here, we report on the identification and characterization of a plant-specific protein, MULTIPOLAR SPINDLE 1 (MPS1), which is involved in spindle organization in meiocytes of Arabidopsis thaliana. The homozygous mps1 mutant exhibits male and female sterility. Light microscopy showed that mps1 mutants produced multiple uneven spores during anther development, most of which aborted in later stages. Cytological analysis showed that chromosome segregation was abnormal in mps1 meiocytes. Immunolocalization showed unequal bipolar or multipolar spindles in mps1 meiocytes, which indicated that aberrant spindles resulted in disordered chromosome segregation. MPS1 encodes a 377-amino-acid protein with putative coiled-coil motifs. In situ hybridization analysis showed that MPS1 is strongly expressed in meiocytes.

Defective in Tapetal development and function 1 is essential for anther development and tapetal function for microspore maturation in Arabidopsis.

In Arabidopsis, the tapetum plays important roles in anther development by providing enzymes for callose dissolution and materials for pollen-wall formation, and by supplying nutrients for pollen development. Here, we report the identification and characterization of a male-sterile mutant, defective in tapetal development and function 1 (tdf1), that exhibits irregular division and dysfunction of the tapetum. The TDF1 gene was characterized using a map-based cloning strategy, and was confirmed by genetic complementation. It encodes a putative R2R3 MYB transcription factor, and is highly expressed in the tapetum, meiocytes and microspores during anther development. Callose staining and gene expression analysis suggested that TDF1 may be a key component in controlling callose dissolution. Semi-quantitative and quantitative RT-PCR analysis showed that TDF1 acts downstream of DYT1 and upstream of AMS and AtMYB103 in the transcriptional regulatory networks that regulate tapetal development. In conclusion, our results show that TDF1 plays a vital role in tapetal differentiation and function.

RUPTURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis.

During microsporogenesis, the microsporocyte (or microspore) plasma membrane plays multiple roles in pollen wall development, including callose secretion, primexine deposition, and exine pattern determination. However, plasma membrane proteins that participate in these processes are still not well known. Here, we report that a new gene, RUPTURED POLLEN GRAIN1 (RPG1), encodes a plasma membrane protein and is required for exine pattern formation of microspores in Arabidopsis (Arabidopsis thaliana). The rpg1 mutant exhibits severely reduced male fertility with an otherwise normal phenotype, which is largely due to the postmeiotic abortion of microspores. Scanning electron microscopy examination showed that exine pattern formation in the mutant is impaired, as sporopollenin is randomly deposited on the pollen surface. Transmission electron microscopy examination further revealed that the primexine formation of mutant microspores is aberrant at the tetrad stage, which leads to defective sporopollenin deposition on microspores and the locule wall. In addition, microspore rupture and cytoplasmic leakage were evident in the rpg1 mutant, which indicates impaired cell integrity of the mutant microspores. RPG1 encodes an MtN3/saliva family protein that is integral to the plasma membrane. In situ hybridization analysis revealed that RPG1 is strongly expressed in microsporocyte (or microspores) and tapetum during male meiosis. The possible role of RPG1 in microsporogenesis is discussed.

Transcription factor AtMYB103 is required for anther development by regulating tapetum development, callose dissolution and exine formation in Arabidopsis.

Downregulation of the transcription factor AtMYB103 using transgenic technology results in early tapetal degeneration and pollen aberration during anther development in Arabidopsis thaliana. This paper describes the functional analysis of the AtMYB103 gene in three knock-out mutants. Two male sterile mutants, ms188-1 and ms188-2, were generated by ethyl-methane sulfonate (EMS) mutagenesis. A map-based cloning approach was used, and ms188 was mapped to a 95.8-kb region on chromosome 5 containing an AtMYB103 transcription factor. Sequence analysis revealed that ms188-1 had a pre-mature stop codon in the AtMYB103 coding region, whereas ms188-2 had a CCT-->CTT base-pair change in the first exon of AtMYB103, which resulted in the replacement of a proline by a leucine residue in the R2R3 domain. The third mutant, an AtMYB103 transposon-tagging line, also showed a male sterile phenotype. Allelism tests indicated that MS188 and AtMYB103 belong to the same locus. Cytological observation revealed defective tapetum development and altered callose dissolution in ms188 plants. Additionally, most of the microspores in mature anthers were degraded and surviving microspores lacked exine. AtMYB103 encoded an R2R3 MYB protein that is predominantly located in the nucleus. Real-time RT-PCR analysis indicated that the callase-related gene A6 was regulated by AtMYB103. Expression of the exine formation gene MS2 was not detected in mutant anthers. These results implicate that AtMYB103 plays an important role in tapetum development, callose dissolution and exine formation in A. thaliana anthers.

[Functional analysis of rice P0491E01 gene regulating anther development].

The rice P0491E01 gene shares high similarity in amino acid sequence with Arabidopsis gene AtDAD1 (DEFECTIVE IN ANTHER DEHISCENCE1) which plays a key role in the biosynthesis of jasmonic acid. In this paper, we investigated the function of this gene in the anther development of rice using RNA interference strategy. An exon fragment of 263bp was cloned from genomic DNA of P0491E01 to construct RNAi vector pP0491RNAi. Then, pP0491RNAi was transformed into O. sativa japonica by Agrobacterium-mediated transformation and ten transgenic plants were obtained. GUS-staining and PCR analysis confirmed that T-DNA was integrated into rice genome. Three of the transgenic plants were male sterile, and the other transgenic plants showed reduced fertility. Cytological observation indicated that anther development in the early stage of male sterile transgenic plants was normal, however, the microspores could not develop into mature pollen grains. Further investigations of the expression of P0491E01 gene in these transgenic lines by RT-PCR revealed that its transcription was significantly reduced. The results suggest that P0491E01 may play an important role during the late stage of anther development.

Identification and genetic mapping of a novel gene involved in pollen development in arabidopsis.

With an aim of the genetic dissection of anther and pollen development, we identified an Arabidopsis mutant line named zy1511 by ethyl-methane sulphonate (EMS) mutagenesis. Genetic analysis indicated that the mutant phenotype was controlled by a single recessive nuclear gene zy1511. Cytological studies of anther at various developmental stages indicated that the mutant anther tapetum did not start degenerating after microspores released from the tetrads. Part of the mutant anther tapetum still existed at late stages of anther development, indicating that mutant tapetum degenerated later than that of the wild-type. So the mutant microspores could not develop into normal pollen grains in the anther. For the further genetic analysis and the map-based cloning of gene zy1511, we have mapped it to a region of 134 kb between molecular markers F25P12 and T8L23 on chromosome 1 using a map-based cloning strategy. As no male sterile genes have been reported in this region, we believe that zy1511 is an undiscovered key gene involved in pollen development.