April 2014, Volume 56 Issue 4, Pages 334每419.

Cover Caption: Ribozymebased CRISPR Technology
About the cover: A chimeric guide RNA (gRNA) is often used to direct Cas9 protein in the CRISPR system to achieve sequence-specified DNA cleavage. Usually gRNA is produced in vitro by phage polymerases, or in vivo by RNA polymerase III. In this issue, Gao and Zhao (pp. 343每349) report a ribozyme-flanked gRNA based design, allowing gRNA to be produced by other RNA polymerases.


Recent advances in plant membrane-bound transcription factor research: Emphasis on intracellular movement  
Author: Pil Joon Seo
Journal of Integrative Plant Biology 2014 56(4): 334每342
Published Online: December 3, 2013
DOI: 10.1111/jipb.12139

Transcription factors constitute numerous signal transduction networks and play a central role in gene expression regulation. Recent studies have shown that a limited portion of transcription factors are anchored in the cellular membrane, storing as dormant forms. Upon exposure to environmental and developmental cues, these transcription factors are released from the membrane and translocated to the nucleus, where they regulate associated target genes. As this process skips both transcriptional and translational regulations, it guarantees prompt response to external and internal signals. Membrane-bound transcription factors (MTFs) undergo several unique steps that are not involved in the action of canonical nuclear transcription factors: proteolytic processing and intracellular movement. Recently, alternative splicing has also emerged as a mechanism to liberate MTFs from the cellular membranes, establishing an additional activation scheme independent of proteolytic processing. Multiple layers of MTF regulation add complexity to transcriptional regulatory scheme and ensure elaborate action of MTFs. In this review, we provide an overview of recent findings on MTFs in plants and highlight the molecular mechanisms underlying MTF liberation from cellular membranes with an emphasis on intracellular movement.

Seo PJ (2014) Recent advances in plant membrane坼bound transcription factor research: Emphasis on intracellular movement. J Integr Plant Biol 56: 334–342. doi: 10.1111/jipb.12139

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          New Technology
Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing
Author: Yangbin Gao and Yunde Zhao
Journal of Integrative Plant Biology 2014 56(4): 343每349
Published Online: December 30, 2013
DOI: 10.1111/jipb.12152

CRISPR/Cas9 uses a guide RNA (gRNA) molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.

Gao Y, Zhao Y (2014) Self坼processing of ribozyme坼flanked RNAs into guide RNAs in vitro and in vivo for CRISPR坼mediated genome editing. J Integr Plant Biol 56: 343–349. doi: 10.1111/jipb.12152

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          Cell and Developmental Biology
Auxin biosynthesis by the YUCCA6 flavin monooxygenase gene in woodland strawberry
Author: Hong Liu, Wei-Fa Xie, Ling Zhang, Victoriano Valpuesta, Zheng-Wen Ye, Qing-Hua Gao and Ke Duan
Journal of Integrative Plant Biology 2014 56(4): 350每363
Published Online: December 20, 2013
DOI: 10.1111/jipb.12150

Auxin has been regarded as the main signal molecule coordinating the growth and ripening of fruits in strawberry, the reference genomic system for Rosaceae. The mechanisms regulating auxin biosynthesis in strawberry are largely elusive. Recently, we demonstrated that two YUCCA genes are involved in flower and fruit development in cultivated strawberry. Here, we show that the woodland strawberry (Fragaria vesca L.) genome harbors nine loci for YUCCA genes and eight of them encode functional proteins. Transcription pattern in different plant organs was different for all eight FvYUCs. Functionality of the FvYUC6 gene was studied in transgenic strawberry overexpressing FvYUC6, which showed typical high-auxin phenotypes. Overexpression of FvYUC6 also delayed flowering and led to complete male sterility in F. vesca. Additionally, specific repression of FvYUC6 expression by RNA interference significantly inhibited vegetative growth and reduced plant fertility. The development of leaves, roots, flowers, and fruits was greatly affected in FvYUC6-repressed plants. Expression of a subset of auxin-responsive genes was well correlated with the changes of FvYUC6 transcript levels and free indole-3-acetic acid levels in transgenic strawberry. These observations are consistent with an important role of FvYUC6 in auxin synthesis, and support a main role of the gene product in vegetative and reproductive development in woodland strawberry.

Liu H, Xie WF, Zhang L, Valpuesta V, Ye ZW, Gao QH, Duan K (2014) Auxin biosynthesis by the YUCCA6 flavin monooxygenase gene in woodland strawberry. J Integr Plant Biol 56: 350–363. doi: 10.1111/jipb.12150

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Cloning and characterization of a potato StAN11 gene involved in anthocyanin biosynthesis regulation
Author: Wang Li, Bing Wang, Man Wang, Min Chen, Jing-Ming Yin, Ghullam Murtaza Kaleri, Rui-Jie Zhang, Tie-Niu Zuo, Xiong You and Qing Yang
Journal of Integrative Plant Biology 2014 56(4): 364每372
Published Online: December 4, 2013
DOI: 10.1111/jipb.12136

Anthocyanins are a class of products of plant secondary metabolism and are responsible for tubers color in potato. The biosynthesis of anthocyanins is a complex biological process, in which multiple genes are involved including structural genes and regulatory genes. In this study, StAN11, a WD40-repeat gene, was cloned from potato cultivar Chieftain (Solanum tuberosum L.). StAN11 (HQ599506) contained no intron and its open reading frame (ORF) was 1,029 bp long, encoding a putative protein of 342 amino acids. In order to verify its role in anthocyanin biosynthesis, StAN11 was inserted behind the CaMV-35S promoter of pCMBIA1304 and the recombination vector was introduced into the potato cultivar Désirée plants by Agrobacterium-mediated transformation. The color of transgenic tuber skin was significantly deepened, compared to the wild-type control, which was highly consistent with the accumulation of anthocyanin and expression of StAN11 in transgenic lines tuber skin. Further analysis on the expression of Flavonone-3-hydroxylase (F3H), Dihydroflavonol reductase (DFR), Anthocyanidin synthase (ANS), and Flavonoid 3-O-glucosyl transferase (3GT) in transgenic plants revealed that only DFR was upregulated. This result suggested that StAN11 regulated anthocyanin biosynthesis in potato by controlling DFR expression and accumulation of anthocyanin could be increased through overexpression of StAN11 in the tubers with the genetic background of anthocyanin biosynthesis.

Li W, Wang B, Wang M, Chen M, Yin JM, Kaleri GM, Zhang RJ, Zuo TN, You X, Yang Q (2014) Cloning and characterization of a potato StAN11 gene involved in anthocyanin biosynthesis regulation. J Integr Plant Biol 56: 364–372. doi: 10.1111/jipb.12136

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Overexpression of OsPIL15, a phytochrome-interacting factor-like protein gene, represses etiolated seedling growth in rice
Author: Jinjun Zhou, Qianqian Liu, Fang Zhang, Yingying Wang, Shiyong Zhang, Huimin Cheng, Lihua Yan, Li Li, Fan Chen and Xianzhi Xie
Journal of Integrative Plant Biology 2014 56(4): 373每387
Published Online: November 26, 2013
DOI: 10.1111/jipb.12137

Phytochrome-interacting factors (PIFs) regulate an array of developmental responses ranging from seed germination to vegetational architecture in Arabidopsis. However, information regarding the functions of the PIF family in monocots has not been widely reported. Here, we investigate the roles of OsPIL15, a member of the rice (Oryza sativa L. cv. Nipponbare) PIF family, in regulating seedling growth. OsPIL15 encodes a basic helix-loop-helix factor localized in the nucleus. OsPIL15-OX seedlings exhibit an exaggerated shorter aboveground part and undeveloped root system relative to wild-type seedlings, suggesting that OsPIL15 represses seedling growth in the dark. Microarray analysis combined with gene ontology analysis revealed that OsPIL15 represses a set of genes involved in auxin pathways and cell wall organization or biogenesis. Given the important roles of the auxin pathway and cell wall properties in controlling plant growth, we speculate that OsPIL15 represses seedling growth likely by regulating the auxin pathway and suppressing cell wall organization in etiolated rice seedlings. Additionally, exposure to red light or far-red light relieved growth retardation and promoted seedling elongation in the OsPIL15-OX lines, despite higher levels of OsPIL15 transcripts under red light and far-red light than in the dark. These results suggest that light regulation of OsPIL15 expression is probably involved in photomorphogenesis in rice.

Zhou J, Liu Q, Zhang F, Wang Y, Zhang S, Cheng H, Yan L, Li L, Chen F, Xie X (2014) Overexpression of OsPIL15, a phytochrome坼interacting factor坼like protein gene, represses etiolated seedling growth in rice. J Integr Plant Biol 56: 373–387. doi: 10.1111/jipb.12137

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          Functional Omics and Systems Biology
Identification, occurrence, and validation of DRE and ABRE Cis坼regulatory motifs in the promoter regions of genes of Arabidopsis thaliana  
Author: Sonal Mishra, Aparna Shukla, Swati Upadhyay, Sanchita, Pooja Sharma, Seema Singh, Ujjal J. Phukan, Abha Meena, Feroz Khan, Vineeta Tripathi, Rakesh Kumar Shukla and Ashok Shrama
Journal of Integrative Plant Biology 2014 56(4): 388每399
Published Online: December 20, 2013
DOI: 10.1111/jipb.12149

Plants posses a complex co-regulatory network which helps them to elicit a response under diverse adverse conditions. We used an in silico approach to identify the genes with both DRE and ABRE motifs in their promoter regions in Arabidopsis thaliana. Our results showed that Arabidopsis contains a set of 2,052 genes with ABRE and DRE motifs in their promoter regions. Approximately 72% or more of the total predicted 2,052 genes had a gap distance of less than 400 bp between DRE and ABRE motifs. For positional orientation of the DRE and ABRE motifs, we found that the DR form (one in direct and the other one in reverse orientation) was more prevalent than other forms. These predicted 2,052 genes include 155 transcription factors. Using microarray data from The Arabidopsis Information Resource (TAIR) database, we present 44 transcription factors out of 155 which are upregulated by more than twofold in response to osmotic stress and ABA treatment. Fifty-one transcripts from the one predicted above were validated using semiquantitative expression analysis to support the microarray data in TAIR. Taken together, we report a set of genes containing both DRE and ABRE motifs in their promoter regions in A. thaliana, which can be useful to understand the role of ABA under osmotic stress condition.

Mishra S, Shukla A, Upadhyay S, Sanchita, Sharma P, Singh S, Phukan UJ, Meena A, Khan F, Tripathi V, Shukla RK, Shrama A (2014) Identification, occurrence and validation of DRE and ABRE cis坼regulatory motifs in the promoter regions of genes of Arabidopsis thaliana. J Integr Plant Biol 56: 388–399. doi: 10.1111/jipb.12149

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          Molecular Physiology
The rice OsV4 encoding a novel pentatricopeptide repeat protein is required for chloroplast development during the early leaf stage under cold stress
Author: Xiaodi Gong, Qianqian Su, Dongzhi Lin, Quan Jiang, Jianlong Xu, Jianhui Zhang, Sheng Teng and Yanjun Dong
Journal of Integrative Plant Biology 2014 56(4): 400每410
Published Online: November 30, 2013
DOI: 10.1111/jipb.12138

Pentatricopeptide repeat (PPR) proteins, characterized by tandem arrays of a 35 amino acid motif, have been suggested to play central and broad roles in modulating the expression of organelle genes in plants. However, the molecular mechanisms of most rice PPR genes remains unclear. In this paper, we isolated and characterized a temperature-conditional virescent mutant, OsV4, in rice (Oryza sativa cultivar Jiahua1 (WT, japonica rice variety)). The mutant displays albino phenotype and abnormal chloroplasts at the three leaf stage, which gradually turns green after the four leaf stage at a low temperature (20 °C). But the mutant always develops green leaves and well-developed chloroplasts at a high temperature (32 °C). Genetic and molecular analyses uncovered that OsV4 encodes a novel chloroplast-targeted PPR protein including four PPR motifs. Further investigations show that the mutant phenotype is associated with changes in chlorophyll content and chloroplast development. The OsV4 transcripts only accumulate to high levels in young leaves, indicating that its expression is tissue-specific. In addition, transcript levels of some ribosomal components and plastid-encoded polymerase-dependent genes are dramatically reduced in the albino mutants grown at 20 °C. These findings suggest that OsV4 plays an important role during early chloroplast development under cold stress in rice.

Gong X, Su Q, Lin D, Jiang Q, Xu J, Zhang J, Teng S, Dong Y (2014) The rice OsV4 encoding a novel pentatricopeptide repeat protein is required for chloroplast development during the early leaf stage under cold stress. J Integr Plant Biol 56: 400–410. doi: 10.1111/jipb.12138

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          Plant Reproduction
Pre- and post-zygotic reproductive isolation between co-occurring Mussaenda pubescens var. alba and M. shikokiana (Rubiaceae)
Author: Shi Chen, Zhonglai Luo and Dianxiang Zhang
Journal of Integrative Plant Biology 2014 56(4): 411每419
Published Online: December 3, 2013
DOI: 10.1111/jipb.12140

Reproductive isolation is a fundamental requirement for speciation and includes several sequential stages. Few studies have determined the relative contributions of pre- and post-zygotic reproductive isolation in plants, especially between relative species with clear differentiation in flower form. To investigate the mechanisms responsible for reproductive isolation in sympatric Mussaenda pubescens var. alba and Mussaenda shikokiana (Rubiaceae) in Guangxi Province, China, we made observations of flowering phenology, patterns of insect visitation, and conducted pollination experiments, including artificial hybridization. The two species had overlapping flowering times and were pollinated by overlapping pollinators; however, their relative importance differed significantly with M. pubescens visited more commonly by bees and M. shikokiana more frequently by butterflies. Using vegetative and floral characters and molecular evidence based on nuclear ribosomal internal and external transcribed spacer regions we detected seven naturally occurring hybrids among a sample of approximately 125 individuals. Hybrids were characterized by morphologies that most closely resembled their maternal parents based on chloroplast evidence. Studies of artificially synthesized and natural hybrids demonstrated that hybrid seed had very low germination rates and naturally occurring hybrids exhibited pollen sterility. Post-zygotic reproductive isolating mechanisms play a primary role in limiting gene exchange between co-occurring species and maintaining species integrity in areas of sympatry.

Chen S, Luo Z, Zhang D (2014) Pre坼and post坼zygotic reproductive isolation between co坼occurring Mussaenda pubescens var. alba and M. shikokiana (Rubiaceae). J Integr Plant Biol 56: 411–419. doi: 10.1111/jipb.12140

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