Zhong-Nan Yang

Development of Genome-Wide DNA Polymorphism Database for Map-Based Cloning of Rice Genes

DNA polymorphism is the basis to develop molecular markers that are widely used in genetic mapping today. A genome-wide rice (Oryza sativa) DNA polymorphism database has been constructed in this work using the genomes of Nipponbare, a cultivar of japonica, and 93-11, a cultivar of indica. This database contains 1,703,176 single nucleotide polymorphisms (SNPs) and 479,406 Insertion/Deletions (InDels), approximately one SNP every 268 bp and one InDel every 953 bp in rice genome. Both SNPs and InDels in the database were experimentally validated. Of 109 randomly selected SNPs, 107 SNPs (98.2%) are accurate. PCR analysis indicated that 90% (97 of 108) of InDels in the database could be used as molecular markers, and 68% to 89% of the 97 InDel markers have polymorphisms between other indica cultivars (Guang-lu-ai 4 and Long-te-pu B) and japonica cultivars (Zhong-hua 11 and 9522). This suggests that this database can be used not only for Nipponbare and 93-11, but also for other japonica and indica cultivars. While validating InDel polymorphisms in the database, a set of InDel markers with each chromosome 3 to 5 marker was developed. These markers are inexpensive and easy to use, and can be used for any combination of japonica and indica cultivars used in this work. This rice DNA polymorphism database will be a valuable resource and important tool for map-based cloning of rice gene, as well as in other various research on rice (http://shenghuan.shnu.edu.cn/ricemarker).

The Putative RNA-Dependent RNA Polymerase RDR6 Acts Synergistically with ASYMMETRIC LEAVES1 and 2 to Repress BREVIPEDICELLUS and MicroRNA165/166 in Arabidopsis Leaf Development

The Arabidopsis thaliana ASYMMETRIC LEAVES1 (AS1) and AS2 genes are important for repressing class I KNOTTED1-like homeobox (KNOX) genes and specifying leaf adaxial identity in leaf development. RNA-dependent RNA polymerases (RdRPs) are critical for posttranscriptional and transcriptional gene silencing in eukaryotes; however, very little is known about their functions in plant development. Here, we show that the Arabidopsis RDR6 gene (also called SDE1 and SGS2) that encodes a putative RdRP, together with AS1 and AS2, regulates leaf development. rdr6 single mutant plants displayed only minor phenotypes, whereas rdr6 as1 and rdr6 as2 double mutants showed dramatically enhanced as1 and as2 phenotypes, with severe defects in the leaf adaxial-abaxial polarity and vascular development. In addition, the double mutant plants produced more lobed leaves than the as1 and as2 single mutants and showed leaf-like structures associated on a proportion of leaf blades. The abnormal leaf morphology of the double mutants was accompanied by an extended ectopic expression of a class I KNOX gene BREVIPEDICELLUS (BP) and high levels of microRNA165/166 that may lead to mRNA degradation of genes in the class III HD-ZIP family. Taken together, our data suggest that the Arabidopsis RDR6-associated epigenetic pathway and the AS1-AS2 pathway synergistically repress BP and MIR165/166 for proper plant development.

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.

AtECB2, a pentatricopeptide repeat protein, is required for chloroplast transcript accD RNA editing and early chloroplast biogenesis in Arabidopsis thaliana

Chloroplast biogenesis is a complex process in higher plants. Screening chloroplast biogenesis mutants, and elucidating their molecular mechanisms, will provide insight into the process of chloroplast biogenesis. In this paper, we obtained an early chloroplast biogenesis mutant atecb2 that displayed albino cotyledons and was seedling lethal. Microscopy observations revealed that the chloroplast of atecb2 mutants lacked an organized thylakoid membrane. The AtECB2 gene, which is highly expressed in cotyledons and seedlings, encodes a pentatricopeptide repeat protein (PPR) with a C-terminal DYW domain. The AtECB2 protein is localized in the chloroplast, and contains a conserved HxEx(n)CxxC motif that is similar to the activated site of cytidine deaminase. The AtECB2 mutation affects the expression pattern of plastid-encoded genes. Immunoblot analyses showed that the levels of photosynthetic proteins decreased substantially in atecb2 mutants. Inspection of all reported plastid RNA editing sites revealed that one editing site, accD, is not edited in atecb2 mutants. Therefore, the AtECB2 protein must regulate the RNA editing of this site, and the dysfunctional AccD protein from the unedited RNA molecules could lead to the mutated phenotype. All of these results indicate that AtECB2 is required for chloroplast transcript accD RNA editing and early chloroplast biogenesis in Arabidopsis thaliana.

WOX11 and 12 Are Involved in the First-Step Cell Fate Transition during de Novo Root Organogenesis in Arabidopsis

Adventitious roots and shoots can be regenerated from wounded or detached plant organs via de novo organogenesis. This study reveals the cellular and molecular framework of de novo root organogenesis from leaf explants of Arabidopsis thaliana and shows that auxin-induced WOX11 expression marks the first-step cell fate transition in this process. De novo organogenesis is a process through which wounded or detached plant tissues or organs regenerate adventitious roots and shoots. Plant hormones play key roles in de novo organogenesis, whereas the mechanism by which hormonal actions result in the first-step cell fate transition in the whole process is unknown. Using leaf explants of Arabidopsis thaliana, we show that the homeobox genes WUSCHEL RELATED HOMEOBOX11 (WOX11) and WOX12 are involved in de novo root organogenesis. WOX11 directly responds to a wounding-induced auxin maximum in and surrounding the procambium and acts redundantly with its homolog WOX12 to upregulate LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) and LBD29, resulting in the first-step cell fate transition from a leaf procambium or its nearby parenchyma cell to a root founder cell. In addition, our results suggest that de novo root organogenesis and callus formation share a similar mechanism at initiation.

A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression

The SET domain-containing protein, pTAC14, was previously identified as a component of the transcriptionally active chromosome (TAC) complexes. Here, we investigated the function of pTAC14 in the regulation of plastid-encoded bacterial-type RNA polymerase (PEP) activity and chloroplast development. The knockout of pTAC14 led to the blockage of thylakoid formation in Arabidopsis (Arabidopsis thaliana), and ptac14 was seedling lethal. Sequence and transcriptional analysis showed that pTAC14 encodes a specific protein in plants that is located in the chloroplast associated with the thylakoid and that its expression depends on light. In addition, the transcript levels of all investigated PEP-dependent genes were clearly reduced in the ptac14-1 mutants, while the accumulation of nucleus-encoded phage-type RNA polymerase-dependent transcripts was increased, indicating an important role of pTAC14 in maintaining PEP activity. pTAC14 was found to interact with pTAC12/HEMERA, another component of TACs that is involved in phytochrome signaling. The data suggest that pTAC14 is essential for proper chloroplast development, most likely by affecting PEP activity and regulating PEP-dependent plastid gene transcription in Arabidopsis together with pTAC12.

Auxin Response Factor17 is essential for pollen wall pattern formation in Arabidopsis

The isolation and characterization of the arf17 mutant uncovers potential roles for auxin in pollen wall pattern formation and pollen tube growth. In angiosperms, pollen wall pattern formation is determined by primexine deposition on the microspores. Here, we show that AUXIN RESPONSE FACTOR17 (ARF17) is essential for primexine formation and pollen development in Arabidopsis (Arabidopsis thaliana). The arf17 mutant exhibited a male-sterile phenotype with normal vegetative growth. ARF17 was expressed in microsporocytes and microgametophytes from meiosis to the bicellular microspore stage. Transmission electron microscopy analysis showed that primexine was absent in the arf17 mutant, which leads to pollen wall-patterning defects and pollen degradation. Callose deposition was also significantly reduced in the arf17 mutant, and the expression of CALLOSE SYNTHASE5 (CalS5), the major gene for callose biosynthesis, was approximately 10% that of the wild type. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that ARF17 can directly bind to the CalS5 promoter. As indicated by the expression of DR5-driven green fluorescent protein, which is an synthetic auxin response reporter, auxin signaling appeared to be specifically impaired in arf17 anthers. Taken together, our results suggest that ARF17 is essential for pollen wall patterning in Arabidopsis by modulating primexine formation at least partially through direct regulation of CalS5 gene expression.

A Genetic Pathway for Tapetum Development and Function in Arabidopsis

In anther development, tapetal cells take part in complex processes, including endomitosis and apoptosis (programmed cell death). The tapetum provides many of the proteins, lipids, polysaccharides and other molecules necessary for pollen development. Several transcription factors, including DYT1, TDF1, AMS, MS188 and MS1, have been reported to be essential for tapetum development and function in Arabidopsis thaliana. Here, we present a detailed cytological analysis of knockout mutants for these genes, along with an in situ RNA hybridization experiment and double mutant analysis showing that these transcription factors form a genetic pathway in tapetum development. DYT1, TDF1 and AMS function in early tapetum development, while MS188 and MS1 are important for late tapetum development. The genetic pathway revealed in this work facilitates further investigation of the function and molecular mechanisms of tapetum development in Arabidopsis.

The tapetal AHL family protein TEK determines nexine formation in the pollen wall

The pollen wall, an essential structure for pollen function, consists of two layers, an inner intine and an outer exine. The latter is further divided into sexine and nexine. Many genes involved in sexine development have been reported, in which the MYB transcription factor Male Sterile 188 (MS188) specifies sexine in Arabidopsis. However, nexine formation remains poorly understood. Here we report the knockout of TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK) leads to nexine absence in Arabidopsis. TEK encodes an AT-hook nuclear localized family protein highly expressed in tapetum during the tetrad stage. Absence of nexine in tek disrupts the deposition of intine without affecting sexine formation. We find that ABORTED MICROSPORES directly regulates the expression of TEK and MS188 in tapetum for the nexine and sexine formation, respectively. Our data show that a transcriptional cascade in the tapetum specifies the development of pollen wall.