Chang-en Tian

Arabidopsis AUXIN RESPONSE FACTOR6 and 8 Regulate Jasmonic Acid Biosynthesis and Floral Organ Development via Repression of Class 1 KNOX Genes

Two mutations in Arabidopsis thaliana, auxin response factor6 (arf6) and arf8, concomitantly delayed the elongation of floral organs and subsequently delayed the opening of flower buds. This phenotype is shared with the jasmonic acid (JA)-deficient mutant dad1, and, indeed, the JA level of arf6 arf8 flower buds was decreased. Among JA biosynthetic genes, the expression level of DAD1 (DEFECTIVE IN ANTHER DEHISCENCE1) was markedly decreased in the double mutant, suggesting that ARF6 and ARF8 are required for activation of DAD1 expression. The double mutant arf6 arf8 also showed other developmental defects in flowers, such as aberrant vascular patterning and lack of epidermal cell differentiation in petals. We found that class 1 KNOX genes were expressed ectopically in the developing floral organs of arf6 arf8, and mutations in any of the class 1 KNOX genes (knat2, knat6, bp and hemizygous stm) partially suppressed the defects in the double mutant. Furthermore, ectopic expression of the STM gene caused a phenotype similar to that of arf6 arf8, including the down-regulation of DAD1 expression. These results suggested that most defects in arf6 arf8 are attributable to abnormal expression of class 1 KNOX genes. The expression of AS1 and AS2 was not affected in arf6 arf8 flowers, and as1 and arf6 arf8 additively increased the expression of class 1 KNOX genes. We concluded that ARF6 and ARF8, in parallel with AS1 and AS2, repress the class 1 KNOX genes in developing floral organs to allow progression of the development of these organs.

Transcriptome analysis of Cymbidium sinense and its application to the identification of genes associated with floral development

BackgroundCymbidium sinense belongs to the Orchidaceae, which is one of the most abundant angiosperm families. C. sinense, a high-grade traditional potted flower, is most prevalent in China and some Southeast Asian countries. The control of flowering time is a major bottleneck in the industrialized development of C. sinense. Little is known about the mechanisms responsible for floral development in this orchid. Moreover, genome references for entire transcriptome sequences do not currently exist for C. sinense. Thus, transcriptome and expression profiling data for this species are needed as an important resource to identify genes and to better understand the biological mechanisms of floral development in C. sinense.ResultsIn this study, de novo transcriptome assembly and gene expression analysis using Illumina sequencing technology were performed. Transcriptome analysis assembles gene-related information related to vegetative and reproductive growth of C. sinense. Illumina sequencing generated 54,248,006 high quality reads that were assembled into 83,580 unigenes with an average sequence length of 612 base pairs, including 13,315 clusters and 70,265 singletons. A total of 41,687 (49.88%) unique sequences were annotated, 23,092 of which were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Gene Ontology (GO) analysis of the annotated unigenes revealed that the majority of sequenced genes were associated with metabolic and cellular processes, cell and cell parts, catalytic activity and binding. Furthermore, 120 flowering-associated unigenes, 73 MADS-box unigenes and 28 CONSTANS-LIKE (COL) unigenes were identified from our collection. In addition, three digital gene expression (DGE) libraries were constructed for the vegetative phase (VP), floral differentiation phase (FDP) and reproductive phase (RP). The specific expression of many genes in the three development phases was also identified. 32 genes among three sub-libraries with high differential expression were selected as candidates connected with flower development.ConclusionRNA-seq and DGE profiling data provided comprehensive gene expression information at the transcriptional level that could facilitate our understanding of the molecular mechanisms of floral development at three development phases of C. sinense. This data could be used as an important resource for investigating the genetics of the flowering pathway and various biological mechanisms in this orchid.

Changes in growth kinetics of stamen filaments cause inefficient pollination in massugu2, an auxin insensitive, dominant mutant of Arabidopsis thaliana

We investigated the physiological and molecular basis of lower fecundity of massugu2 (msg2), which is a dominant mutant of an auxin primary response gene, IAA19, in Arabidopsis thaliana. By measuring the length of all stamens and pistils in inflorescences and the reference growth rate of pistils, we constructed growth curves of pistils and stamens between stages 12 and 15 of flower development. Pistil growth was found to consist of a single exponential growth, while stamen growth consisted of three exponential phases. During the second exponential phase, the growth rate of stamen filaments was approximately 10 times greater than the growth rates in the other two phases. Consequently, stamens whose growth was initially retarded grew longer than the pistil, putting pollen grains on the stigma. msg2-1 stamens, on the other hand, exhibited a less obvious growth increase, resulting in less frequent contact between anthers and stigma. MSG2 was expressed in the stamen filaments and its expression almost coincided with the second growth phase. Stamen filaments appeared to elongate by cell elongation rather than cell division in the epidermal cell file. Considering that MSG2 is likely to be a direct target of the auxin F-box receptors, MSG2 may be one of the master genes that control the transient growth increase of stamen filaments.

AtIQM1, a novel calmodulin-binding protein, is involved in stomatal movement in Arabidopsis

We recently identified a novel IQ motif-containing protein family, IQM, which shares sequence homology with a pea heavy metal-induced protein 6 and a ribosome inactivating protein, trichosanthin. Distinct expression patterns for each gene suggest that each IQM family member may play a different role in plant development and response to environmental cues. However functions of the IQM family members remain to be analyzed. IQM1 bound with calmodulin 5 (CaM5) in yeast two-hybrid assay via its IQ-motif. The CaM binding was Ca2+-independent in vitro, and was also observed in bimolecular fluorescence complementation analyses in onion epidermal cells. IQM1 was found to express strongly in guard cells and the cortex of roots. The T-DNA insertion mutants of IQM1 displayed a smaller stomatal aperture, a decreased water loss rate and a shorter primary root. Moreover, iqm1 did not change its stomatal aperture when treated with light, dark, ABA and chitin obviously. Microarray analyses showed that 243 and 28 genes were up- and down-regulated by more than twofold in iqm1-1, respectively. Interesting, 34 of 117 and 7 of 30 chitin-responsive transcriptional factor and ubiquitin ligase genes were up-regulated, respectively. Stomatal guard cells of iqm1-1 also showed enhanced expression of genes involved in production and signaling of reactive oxygen species (ROS). Consistently, increased ROS level was observed in the iqm1 guard cells.

Gene cloning and characterization of a novel laccase from the tropical white-rot fungus Ganoderma weberianum TZC-1

The novel laccase gene GwLac1 was cloned from Ganoderma weberianum TZC-1 by reverse transcriptase PCR with degenerate primers on the basis of conserved copper-binding regions of known laccases and the rapid amplification of cDNA ends technique. GwLac1 contains 9 introns and its open reading frame is 1566 bp long. The coding domain sequence of GwLac1 was cloned into the vector pPICZB and expressed in Pichia pastoris strain GS115, resulting in the highest yield of laccase, 2.26 U/mL, when the transformants had been cultivated at 20°C for 8 days. The molecular mass of recombinant GwLAC1 was a little more than 50 kDa as estimated by SDS-PAGE and exhibited catalytic properties with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a substrate. rGwLAC1 was stable at pH of 5.0–7.0 and at temperatures <45°C. The pH and temperature optima and Km of the enzyme for ABTS oxidation were 2.2, 35°C and 85.5 μM, respectively.

Disruption of IQM5 delays flowering possibly through modulating the juvenile-to-adult transition

IQ motif-containing protein 5 (IQM5) is the fifth member of the IQM family in Arabidopsis, which is an IQ motif-containing family. However, no functional characterizations have been performed using the IQM5 protein in Arabidopsis thaliana. Here, we showed that the IQM5 gene is involved in the regulation of flowering in Arabidopsis. The IQM5 mutants iqm5-1 and iqm5-2 displayed a later-flowering phenotype under both long-day and short-day conditions when compared with wild type. After gibberellic acid or paclobutrazol (PAC) treatments, iqm5-1 and iqm5-2 displayed similar flowering phenotype or PAC sensitivity to wild type. Meanwhile, iqm5-1 and iqm5-2 showed the same flowering ratio as wild type after a vernalization treatment. In addition, disrupting IQM5 increased the transcript level of FLOWERING LOCUS C in both shoot apical meristems and leaves, and decreased that of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 in leaves, but did not change the expression of other key genes in the flowering time-related pathway. In addition, the number of abaxial trichomes in both iqm5-1 and iqm5-2 is lower than that in the wild type. Thus, disruption of IQM5 delays flowering possibly through modulating the juvenile-to-adult transition.

Erratum to: Sequence and expression analysis of the Arabidopsis IQM family

The online version of the original article can be found underdoi:10.1007/s11738-009-0398-9.Y. Zhou J. Duan (&)South China Botanical Garden,Chinese Academy of Sciences,510650 Guangzhou, Chinae-mail: duanj@scib.ac.cnY. Zhou Y. Chen C. Tian (&)Guangzhou Key Laboratory for Functional Study on PlantStress-Resistant Genes, Guangzhou University,510006 Guangzhou, Chinae-mail: changentian@yahoo.com.cnY. ZhouGraduate School of the Chinese Academy of Sciences,100039 Beijing, ChinaY. Zhou C. TianSchool of Life Science, Guangzhou University,510006 Guangzhou, ChinaK. T. YamamotoDepartment of Biological Sciences, Faculty of Science,Hokkaido University, Sapporo 060-0810, Japan

Arabidopsis IQM4, a Novel Calmodulin-Binding Protein, is Involved with Seed Dormancy and Germination in Arabidopsis

Seed dormancy and germination are regulated by complex mechanisms controlled by diverse hormones and environmental cues. Abscisic acid (ABA) promotes seed dormancy and inhibits seed germination and post-germination growth. Calmodulin (CaM) signals are involved with the inhibition of ABA during seed germination and seedling growth. In this study, we showed that Arabidopsis thaliana IQM4 could bind with calmodulin 5 (CaM5) both in vitro and in vivo, and that the interaction was the Ca2+-independent type. The IQM4 protein was localized in the chloroplast and the IQM4 gene was expressed in most tissues, especially the embryo and germinated seedlings. The T-DNA insertion mutants of IQM4 exhibited the reduced primary seed dormancy and lower ABA levels compared with wild type seeds. Moreover, IQM4 plays key roles in modulating the responses to ABA, salt, and osmotic stress during seed germination and post-germination growth. T-DNA insertion mutants exhibited ABA-insensitive and salt-hypersensitive phenotypes during seed germination and post-germination growth, whereas IQM4-overexpressing lines had ABA- and osmotic-hypersensitive, and salt-insensitive phenotypes. Gene expression analyses showed that mutation of IQM4 inhibited the expression of ABA biosynthetic genes NCED6 and NCED9, and seed maturation regulators LEC1, LEC2, ABI3, and ABI5 during the silique development, as well as promoted the expression of WRKY40 and inhibited that of ABI5 in ABA-regulated seed germination. These observations suggest that IQM4 is a novel Ca2+-independent CaM-binding protein, which is positively involved with seed dormancy and germination in Arabidopsis.