Effect of phytohormones on fiber initiation of cotton ovule

In order to study the effect of phytohormones on cotton fiber initiation, contents of four endogenous phytohormones and activities of four related enzymes in ovules (in vivo) of a fuzzless–lintless mutant (fl) and its wild-type (FL) line were measured from 4 days before anthesis (day −4) to 4 days after anthesis (day 4). The results showed that contents of indole-3-acetic acid, gibberellic acid (GA), and zeatin riboside in fl ovules were lower than those in FL ovules. Therefore, indole-3-acetic acid, GA, and zeatin riboside were thought to be the promoters of fiber initiation. Although abscisic acid (ABA) content in fl ovule was slightly higher than that in FL ovule on day 0, which might imply that ABA inhibited fiber initiation. Fiber initiation could also be influenced by enzyme through regulating synthesis and degradation of related phytohormones since fl ovules were significantly higher in activities of indole-3-acetic acid oxidase, cytokinin oxidase and peroxidase, but lower in activity of tryptophan synthetase than those in FL ovules. To test the above hypothesis, exogenous plant growth regulators were also applied for the culture of ovules from fl and FL in vitro. When no regulators were added, no fiber was induced on fl ovule, but a few fibers were induced in FL ovule. Higher total fiber units (TFU) were observed when indole-3-acetic acid and gibberellic acid (GA₃) were applied either separately or in combination to media. TFU did not increased with zeatin riboside alone, but the highest TFU was achieved when zeatin riboside was applied together with indole-3 acetic acid and GA₃, which implied that fiber initiation could be promoted by them as additive.     

Functional analysis of cotton orthologs of GA signal transduction factors GID1 and SLR1

Gibberellic acid (GA) is both necessary and sufficient to promote fiber elongation in cultured fertilized ovules of the upland cotton variety Coker 312. This is likely due to the temporal and spatial regulation of GA biosynthesis, perception, and subsequent signal transduction that leads to alterations in gene expression and morphology. Our results indicate that the initiation of fiber elongation by the application of GA to cultured ovules corresponds with increased expression of genes that encode xyloglucan endotransglycosylase/hydrolase (XTH) and expansin (EXP) that are involved in promoting cell elongation. To gain a better understanding of the GA signaling components in cotton, that lead to such changes in gene expression, two GA receptor genes (GhGID1a and GhGID1b) and two DELLA protein genes (GhSLR1a and GhSLR1b) that are orthologous to the rice GA receptor (GID1) and the rice DELLA gene (SLR1), respectively, were characterized. Similar to the GA biosynthetic genes, expression of GhGID1a and GhGID1b is under the negative regulation by GA while GA positively regulates GhSLR1a. Recombinant GST-GhGID1s showed GA-binding activity in vitro that was augmented in the presence of GhSLR1a, GhSLR1b, or rice SLR1, indicating complex formation between the receptors and repressor proteins. This was further supported by the GA-dependent interaction of these proteins in yeast cells. Ectopic expression of the GhGID1a in the rice gid1-3 mutant plants rescued the GA-insensitive dwarf phenotype, which demonstrates that it is a functional GA receptor. Furthermore, ectopic expression of GhSLR1b in wild type Arabidopsis led to reduced growth and upregulated expression of DELLA-responsive genes.     

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.     

棉纤维细胞发育过程中钙离子内流和钙依赖蛋白激酶活性的动态变化

随着大规模棉花纤维细胞发育的表达谱分析以及系统性生理和生化研究,维持纤维细胞快速伸长的机制也逐渐被阐明,超长链脂肪酸和植物激素乙烯在该过程中发挥重要作用．本研究检测到在棉花纤维发育初期细胞外钙离子内流增加．棉花cDNA芯片及QRT．PCR的数据均显示,CPKl,CPK32和CRK5的表达在纤维伸长期显著升高．系统研究发现,随着纤维细胞的伸长,CPK总活性显著增加,在开花后10～15天达到最高,提示CPK类激酶在纤维发育过程中扮演重要角色．体外胚珠培养加入CPK的抑制剂TFP或w．7导致纤维不能伸长．在体外胚珠培养中加入外源的乙烯或者超长链脂肪酸可以刺激钙离子的瞬时内流和CPK的活性．因此推断,乙烯或者超长链脂肪酸通过促进CPK的活性增强钙离子内流,从而调控纤维细胞的发育．     

Alternative pathways of tobacco placental development: time of commitment and analysis of a mutant

Previous work from our lab identified mutants, Mgr3 and Mgr9, of tobacco (Nicotiana tabacum) that produced unusual elongated green outgrowths from placentae in vivo. Similarly appearing stigmatoid growths were described developing from some in vitro cultures of excised placentae of tobacco (Hicks and McHughen, 1974, 1977). Here we report a developmental analysis and comparison of the unusual stigmatoid outgrowths seen in in vitro cultures of wild-type and mutant placentae, as well as the green outgrowths seen in vivo in the mutants. The growths produced by wild-type and mutant placental cultures in vitro, and the growths produced by the mutants in vivo, are identified as abnormal stigmas and styles. Wild-type in vitro placental cultures also produce outgrowths identified as homologs of whole carpels. Carpel fusion is not required for differentiation of stigma, style, transmitting tract, vascular traces, ovary, and ovules in these structures. The type and extent of stigmatoid growth production depends upon the age of the explant at excision and culture initiation. Before ovule primordium initiation, few growths are seen in culture; for a short window of time afterward, the primordia are competent to give rise to stigmatoid and carpelloid growths when cultured. After commitment to ovule development occurs, the primordia produce only ovules when cultured. The behavior of the mutant placental cultures is dimorphic. Explants from early stages behave similarly to wild-type when cultured, but differences between wild-type and the mutant behaviors in culture arise at the time when the stigmatoid growths begin to appear in vivo in the mutants. These results imply that ovule primordia pass through stages of distinct sequential restrictions of fate, first to growth as gynoecia, and then second to growth as ovules. The mutant strains described here perturb the commitment to differentiation as ovules.     

Cotton fibers can undergo cell division

Ovular culture was used to determine the cell cycle aspects of cotton fiber cells. Each ovule (Gossypium hirsutum, cultivar, MD51 ne) grown under the conditions used has ~10 000 fiber cells at 4 d postanthesis. About 25% of these cells divide when ovules are cultured at 34C. Mitosis occurs after fiber cells differentiate, producing multicelled fibers. The basal and tip cells of multicelled fibers have the same characteristics as the polar ends of single-celled fibers. Most cell division occurs in ovules cultured at 2-3 d postanthesis. Multicelled fibers are rare in ovules cultured at 1 d postanthesis and absent if cultured at 7 d postanthesis. No multicelled fibers are detectable on ovules sampled from the plant regardless of age. Fiber cell division occurs in the absence of exogenous hormones. The addition of IAA and GA3 to the medium lowers the frequency of multicelled fibers. IAA alone further reduces their frequency, while GA3 by itself has no effect. The number of fiber cells per cultured ovule ranges between 9462 and 11 087 and is not significantly different from the 9892 seen in the plant at 4 d postanthesis. These findings show that a subpopulation of fiber cells, fully differentiated in appearance, retain cell cycle functions up to 4 d postanthesis.     

The role of phytohormones in cotton fiber development

Since 1974, when Beasley and Ting discovered that fertilized ovules of cotton can be cultured in media supplemented with GA along with auxin, the effect of all types of phytohormones on fiber development has been widely studied. Many phytohormones, including GA, IAA, brassinosteroid (Br), ABA, ethylene (Et), and cytokinins (Ck), all have been demonstrated to play important roles during cotton fiber development. In recent years, the rapid development of genomic analysis and the accumulation of high-quality cotton ESTs allowed us to probe phytohormonal gene expression during fiber development. Many phytohormonal genes, including GA-, IAA-, ABA-, Br-, Et-, and Ck-related genes, participating in phytohormone biosynthesis pathways and signal transduction pathway accumulated in the process of cotton fiber development.     

棉花胚珠纤维发育的研究

将未受精的棉花胚珠漂浮培养在加有不同植物激素的BT培养基上,培养48小时或96小时后,用扫描电镜观察纤维发育情况,以及测定胚珠内IAA氧化酶活性变化及内源ABA的含量变化,并和同一时期的大田生长的胚珠进行比较。结果表明:IAA+GA_3是最佳激素组合。在这种激素组合的培养基中培养的未受精胚珠,在纤维发育、酶活性变化等方面,均与大田生长的胚珠相似。这一激素组合还能抑制离体胚珠内源ABA的增长,但同一时期的大田生长的胚珠,其内源ABA含量却相对要高。     

Developmental profiles of wing imaginal discs of flügellos(fl), a wingless mutant of the silkworm, Bombyx mori

 Mutations at the flügellos (fl) locus in Bombyx mori give rise to wingless pupae and moths. To understand the developmental steps responsible for the fl wing defect, we compared the morphological changes and protein synthesis profiles between fl and wild-type (WT) wing discs during larval development. Morphologically, the four wing discs in the fl homozygote larva developed normally at least until the fourth instar, but they were slightly smaller than those of the WT. After the last larval ecdysis, wing epithelial invagination and tracheal migration into the lacunar spaces evidently occurred in the WT wing discs. However, there was no apparent morphological change in fl discs through the fifth instar. The fl wing discs cultured in medium containing 20-hydroxyecdysone (20E) did not grow and develop, although the WT wing discs extended and differentiated under the same conditions. A comparison of protein synthesis in the wing discs revealed that several bands were differentially expressed between the fl and WT. A 41-kDa band expressed abundantly from larval to pharate pupal stages in the WT wing discs was rarely observed in fl discs. Furthermore, in vitro culture studies showed that the 41-kDa protein was induced by 20E and specifically synthesized in WT wing discs after the wandering stage, but not in fl discs. The wing-specific protein synthesis and morphogenesis in fl wing discs may be blocked due to aberrant expression of the fl gene. Received: 6 November 1996 / Accepted: 5 February 1997     

棉纤维蔗糖合酶基因SS3上游调控序列的克隆及其表达分析

棉纤维蔗糖合酶基因SS3在棉纤维发育过程中起着重要作用.采用YADE技术克隆了该基因5′上游1717bp的调控区,该调控区含有典型的启动子核心元件TATA box ,以及TATC box、G box、GCN4 -motif、Prolamin box、Skn 1 likemotif、TCA element、HSE和O2 site等各种顺式调控元件和其他一些反应元件.将此序列和报告基因GUS融合在烟草、棉花中表达.组织化学分析结果显示棉花SuSyR序列启动GUS基因在烟草的子房、胎座、种子以及在棉花花蕾与棉铃中表达.在棉花花蕾蕾长为3mm、6mm、9mm和15mm花蕾中表达主要存在于雄蕊及雄蕊管、胎座等器官;在棉铃中,1DPA棉铃的花柱、花药、子房及胚珠中出现了蓝色,6DPA棉铃的子房及胚珠被染成蓝色,在2 0DPA的棉铃中蓝色只出现在胚珠及其纤维中、在胚珠中只有珠心被染成蓝色,在4 0DPA胚珠中只有纤维呈蓝色.研究结果揭示,棉花的SuSyR调控序列启动GUS基因主要在子房、胚珠和纤维等器官和主叶脉、茎微管束等输导组织中表达,在棉花中尤为明显,表明棉纤维蔗糖合酶基因SS3除参与棉花蕾铃发育、纤维素的合成外,还参与了光合产物的运输与分配过程.     

Independent control of fiber development and nitrate reduction in cultured cotton ovules

Several lines of evidence implicate ammonium as an important factor in the growth and development of cotton (Gossypium hirsutum L.) ovules cultured in vitro. For example, ovules cultured at 28 C require indoleacetic acid (IAA) and either ammonium or gibberellic acid (GA₃) in the medium for fiber development, whereas ovules cultured at 34 C require only IAA. Because of this effect of ammonium supply, it seemed possible that hormones or increased temperature were also promoting the availability of reduced nitrogen by induction of increased nitrate reductase activity in the ovules. This possibility was tested.     

Chronology of the differentiation of cotton (Gossypium hirsutum L.) fiber cells

Cotton fibers are single elongated cells that develop from epidermal cells of the ovule. The chronology of fiber differentiation was investigated using cultured ovules. Epidermal cells differentiate into fiber cells approx. 3 d before anthesis. When ovules were cultured on a defined medium, fiber growth could be initiated on ovules any time between 2 d preanthesis and the time of anthesis by adding indole-3-acetic acid and gibberellic acid to the medium. In the absence of phytohormones, fibers did not grow, and when ovules between 2 d preanthesis and anthesis were cultured without hormones past the day of anthesis and hormones then added, most ovules failed to produce fibers. The results define the timing of fiber differentiation from epidermal cells, and also define a window of time when differentiated cells are capable of further development. During this window, fiber cells are latent awaiting appropriate stimulation which in the intact plant is apparently associated with anthesis.Abbreviations GA₃ gibberellic acid - IAA indole-3-acetic acid