Genes expressed in sugarcane maturing internodal tissue

To explore gene expression during sugarcane culm maturation, we performed a partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases, while 111 matched uncharacterised plant expressed sequence tags (ESTs) only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. This research has provided a framework for functional gene analysis in sugarcane sucrose-accumulating tissues.     

ISOLATION AND CHARACTERIZATION OF PHASE-SPECIFIC COMPLEMENTARY DNAs FROM SPOROPHYTES AND GAMETOPHYTES OF PORPHYRA PURPUREA (RHODOPHYTA) USING SUBTRACTED COMPLEMENTARY DNA LIBRARIES1

The red alga Porphyra purpurea (Roth) C. Agardh has a life cycle that alternates between shell-boring, filamentous sporophytes and free-living, foliose gametophytes. The significant morphological differences between these two phases suggest that many genes should be developmentally regulated and expressed in a phase-specific manner. In this study, we prepared and screened subtracted complementary DNA (cDNA) libraries specific for the sporophyte and gametophyte of P. purpurea. This involved the construction of cDNA libraries from each phase, followed by the removal of common clones through subtractive hybridization. Sampling of the subtracted libraries indicated that 8–10% of the recombinant colonies in each library were specific for the appropriate phase. Of 20 putative phase-specific cDNAs selected from each subtracted library, eight unique clones were obtained for the sporophyte and seven for the gametophyte. After confirming their phase-specificities by hybridization to gametophyte and sporophyte messenger RNA, these 15 phase-specific cDNAs were sequenced, and the deduced amino acid sequences were used to search protein databanks. Two proteins encoded by the sporophyte-specific cDNAs and two by the gametophyte-specific cDNAs were identified by their similarity to databank entries.     

The cotton kinesin-like calmodulin-binding protein associates with cortical microtubules in cotton fibers

Microtubules in interphase plant cells form a cortical array, which is critical for plant cell morphogenesis. Genetic studies imply that the minus end-directed microtubule motor kinesin-like calmodulin-binding protein (KCBP) plays a role in trichome morphogenesis in Arabidopsis. However, it was not clear whether this motor interacted with interphase microtubules. In cotton (Gossypium hirsutum) fibers, cortical microtubules undergo dramatic reorganization during fiber development. In this study, cDNA clones of the cotton KCBP homolog GhKCBP were isolated from a cotton fiber-specific cDNA library. During cotton fiber development from 10 to 21 DPA, the GhKCBP protein level gradually decreases. By immunofluorescence, GhKCBP was detected as puncta along cortical microtubules in fiber cells of different developmental stages. Thus our results provide evidence that GhKCBP plays a role in interphase cell growth likely by interacting with cortical microtubules. In contrast to fibers, in dividing cells of cotton, GhKCBP localized to the nucleus, the microtubule preprophase band, mitotic spindle, and the phragmoplast. Therefore KCBP likely exerts multiple roles in cell division and cell growth in flowering plants.     

Identification and characterization of differential gene expression in the mesocarp and kernel of oil palm nuts using suppression subtractive hybridization

Suppression subtracted hybridization (SSH) and dot blotting were used to identify differential gene expression in the mesocarp and kernel of oil palm nuts. The different types of nut tissue show differences in fatty acid anabolism and the synthesis of other important compounds. In total, 302 clones from forward SSH libraries and 238 clones from reverse SSH libraries were identified following differential screening, respectively. Among these, 120 clones from the forward SSH library and 81 clones from the reverse SSH library, showed tenfold or more differential expression levels, and were sequenced. Sequence analysis revealed that 76 clones (28 from the forward SSH library and 48 from the reverse SSH library) represent non-redundant cDNA inserts. The differential expression of 39 subset genes in the two different tissues was further confirmed by RT-PCR analysis. Functionally annotated blasting against the GenBank non-redundant protein database classified all 76 candidate genes into six categories, according to their putative functions. Interestingly, our results show that a group of significantly differentially expressed genes are involved in processes associated with oil palm nut maturation, such as the synthesis of medium-chain saturated fatty acids and phytic acid, nut development, and stress/defense responses. This study describes some relationships between gene expression and metabolic pathways in mature oil palm nuts, and contributes to our understanding of oil palm nut ESTs.     

Analysis of xyloglucan endotransglycosylase/hydrolase (XTH) genes from allotetraploid (Gossypium hirsutum) cotton and its diploid progenitors expressed during fiber elongation

Multiple cellular pathways have been shown to be involved during fiber initiation and elongation stages in the cultivated allotetraploid cotton (Gossypium hirsutum). The cell wall enzymes xyloglucan endotransglycosylase/hydrolases (XTH) have been reported to be associated with the biosynthesis of the cell wall and the growth of cotton fibers, probably regulating the plasticity of the primary cell wall. Among various cotton fiber cDNAs found to be preferentially expressed in cotton fibers, a xyloglucan endotransglycosylase (XTH) cDNA was significantly up-regulated during the elongation stage of cotton fiber development. In the present study, we isolated and characterized genomic clones encoding cotton XTH from cultivated cotton (Gossypium hirsutum) and its diploid progenitors (Gossypium arboreum and Gossypium raimondii), designated GhXTH1-1, GhXTH1-2, GaXTH1 and GrXTH, respectively. In addition, we isolated and characterized, by in silico methods, the putative promoter of XTH1 from Gossypium hirsutum. Sequence analysis revealed more than 50% homology to XTH's at the protein level. DNA gel blot hybridization indicated that at least two copies of GhXTH1 are present in Gossypium hirsutum whereas the diploid progenitor species Gossypium arboreum and Gossypium raimondii has only a single copy. Quantitative real-time PCR and high-resolution melting experiments indicated that in Gossypium hirsutum cultivars, in cotton fibers during early stages of fiber elongation specifically expressing only the GhXTH1-1 gene and expression levels of GhXTH1-1 in fibers varies among cultivars differing in fiber percentage and fiber length.     

Role of GhHDA5 in H3K9 deacetylation and fiber initiation in Gossypium hirsutum

Cotton fibers are single‐celled trichomes that initiate from the epidermal cells of the ovules at or before anthesis. Here, we identified that the histone deacetylase (HDAC ) activity is essential for proper cotton fiber initiation. We further identified 15 HDAC s homoeologs in each of the A‐ and D‐subgenomes of Gossypium hirsutum . Few of these HDAC homoeologs expressed preferentially during the early stages of fiber development [?1, 0 and 6 days post‐anthesis (DPA )]. Among them, GhHDA 5 expressed significantly at the time of fiber initiation (?1 and 0 DPA). The in vitro assay for HDAC activity indicated that GhHDA 5 primarily deacetylates H3K9 acetylation marks. Moreover, the reduced expression of GhHDA 5 also suppresses fiber initiation and lint yield in the RNA interference (RNA i) lines. The 0 DPA ovules of GhHDA 5 RNA i lines also showed alterations in reactive oxygen species homeostasis and elevated autophagic cell death in the developing fibers. The differentially expressed genes (DEG s) identified through RNA ‐seq of RNA i line (DEP 12) and their pathway analysis showed that GhHDA 5 modulates expression of many stress and development‐related genes involved in fiber development. The reduced expression of GhHDA 5 in the RNA i lines also resulted in H3K9 hyper‐acetylation on the promoter region of few DEG s assessed by chromatin immunoprecipitation assay. The positively co‐expressed genes with GhHDA 5 showed cumulative higher expression during fiber initiation, and gene ontology annotation suggests their involvement in fiber development. Furthermore, the predicted protein interaction network in the positively co‐expressed genes indicates HDA 5 modulates fiber initiation‐specific gene expression through a complex involving reported repressors.     

Genes expression analyses of sea-island cotton (Gossypium barbadense L.) during fiber development

Sea-island cotton (Gossypium barbadense L.) is one of the most valuable cotton species due to its silkiness, luster, long staples, and high strength, but its fiber development mechanism has not been surveyed comprehensively. We constructed a normalized fiber cDNA library (from −2 to 25 dpa) of G. barbadense cv. Pima 3-79 (the genetic standard line) by saturation hybridization with genomic DNA. We screened Pima 3-79 fiber RNA from five developmental stages using a cDNA array including 9,126 plasmids randomly selected from the library, and we selected and sequenced 929 clones that had different signal intensities between any two stages. The 887 high-quality expressed sequence tags obtained were assembled into 645 consensus sequences (582 singletons and 63 contigs), of which 455 were assigned to functional categories using gene ontology. Almost 50% of binned genes belonged to metabolism functional categories. Based on subarray analysis of the 887 high-quality expressed sequence tags with 0-, 5-, 10-, 15-, and 20-dpa RNA of Pima 3-79 fibers and a mixture of RNA of nonfiber tissues, seven types of expression profiles were elucidated. Furthermore our results showed that phytohormones may play an important role in the fiber development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

利用mRNA荧光差异显示技术规模化筛选棉纤维特异表达基因

以陆地棉（Gossypium hirsutum L.）品种TM-1开花后9d、21d、27d三个不同发育时期的棉花纤维为材料,利用mRNA荧光差异显示 (FDD) 技术,筛选到109个差异显示的cDNA片段。在此基础上,结合两轮反Northern杂交筛选和Northern杂交分析,获得了多个仅在棉花纤维细胞中特异表达或在纤维中优先表达基因的cDNA片段,序列测定和数据库搜索分析表明,这些cDNA片段中的多数还未有报道。本工作为克隆上述基因的全长cDNA,并进一步研究它们在棉纤维发育中的功能奠定了基础。     

The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation

Single-celled cotton fiber (Gossypium hirsutum) provides a unique experimental system to study cell elongation. To investigate the role of the actin cytoskeleton during fiber development, 15 G. hirsutum ACTIN (GhACT) cDNA clones were characterized. RNA gel blot and real-time RT-PCR analysis revealed that GhACT genes are differentially expressed in different tissues and can be classified into four groups. One group, represented by GhACT1, is expressed predominantly in fiber cells and was studied in detail. A 0.8-kb GhACT1 promoter sufficient to confirm its fiber-specific expression was identified. RNA interference of GhACT1 caused significant reduction of its mRNA and protein levels and disrupted the actin cytoskeleton network in fibers. No defined actin network was observed in these fibers and, consequently, fiber elongation was inhibited. Our results suggested that GhACT1 plays an important role in fiber elongation but not fiber initiation.