Comparative proteomic analysis reveals the mechanisms governing cotton fiber differentiation and initiation
Graphical abstract
Highlights
► We compared the ovule proteomes of wild-type and fiberless mutant (fl) during fiber differentiation and initiation in cotton. ► The fl proteome is consistent with increased ROS generation, decreased stress response and lowered ROS scavenging capability. ► ROS homeostasis may be a central regulatory mechanism for cotton fiber morphogenesis. ► Post-transcriptional and post-translational regulation may be pivotal in this process. ► Carbohydrate metabolism, starch accumulation and the content of some carbohydrates were lower in fl mutant than in WT.
Introduction
Cotton fibers are single-celled seed trichomes that differentiate from individual epidermal cells of the ovule integument. Fiber cell differentiation usually occurs 2 to 3 days before anthesis [1], although prefiber cells can be easily distinguished from non-fiber cells as early as 16 h pre-anthesis by SEM [2]. Cotton fibers undergo four distinctive, but overlapping, developmental stages: 1) initiation, 2) elongation, 3) cellulose biosynthesis, and 4) maturation [3], [4], [5]. Although all seed epidermal cells have the potential to become seed hairs, only about 30% eventually develop into spinnable fibers [3], [5], [6], and fiber yield is largely determined by the number of fibers on each ovule surface. The molecular mechanisms controlling fiber differentiation and initiation remain largely unknown. The elucidation of these underlying molecular mechanisms would provide new knowledge of plant cell determination and morphogenesis, and the identification of the proteins involved in these processes would provide candidate genes for the genetic improvement of cotton fiber quality and yield.
Trichome development has been better studied in Arabidopsis than in cotton (Gossypium sp.), but similar genes may be involved in both. In Arabidopsis, epidermal cell patterning is controlled by a multimeric complex comprising TTG1 (WD40), GL3 and/or EGL3 (bHLH), and GL1 (R2R3-MYB), which induces the expression of GL2, a homeodomain-leucine zipper protein [7]. Four TTG1-like genes have been cloned from cotton, and two of these restored trichome formation in ttg1 mutant Arabidopsis plants [8]. Six MYB genes (GhMYB1–6) have been cloned from Gossypium hirsutum [9]. One MYB gene from G. arboreum (GaMYB2) rescued trichome formation in gl1 mutant Arabidopsis plants, and its ectopic expression in Arabidopsis induced trichome formation from Arabidopsis seed epidermis [10]. Another MYB gene from G. hirsutum (GhMYB109) was shown to be expressed specifically in fiber initials and elongating fibers. Antisense-mediated suppression of GhMYB109 resulted in a substantial reduction in fiber length [11]. GhMYB25 is thought to be expressed predominately in ovules and fiber initials [12], [13]. Moreover, an EST similar to AtCPC that acts as an inhibitor of trichome development in Arabidopsis, has been identified in 1 DPA fiber initial [14]. These findings indicate that the genes and pathways involved in the determination of epidermis cell fate are similar between cotton seeds and Arabidopsis leaf trichomes.
In cotton, sucrose synthase (SS3) activity was found to be dramatically reduced in a fiberless seed mutant, whereas it accumulated in the basal areas of initiating fiber cells in wild-type (WT) plants. The suppression of SS3 produces adverse effects on fiber cell initiation and elongation [15], [16]. Recent large-scale transcriptome analyses of − 3, 0, and + 3 DPA immature cotton ovules [17], and of 1 DPA fiber initials [14] revealed genes involved in the molecular mechanisms of fiber differentiation, initiation, and expansion, including transcription factors analogous to those in Arabidopsis for trichome development. It appears that phytohormonal and Ca2+-signaling pathways are involved; however, most of the identified genes have not yet been functionally characterized. Furthermore, cotton functional genomics, facilitated by large-scale expressed sequence tag (EST) sequencing followed by microarray analyses, have focused mostly on fiber elongation and secondary cell wall synthesis [18], [19], [20], [21], [22], [23], [24].
As proteins are direct executors of most cellular functions and processes, proteomics provides a global and integrated view of cellular processes and networks. It also extends our knowledge from gene expression to the metabolite level and finally to phenotype expression. Proteome analyses based upon 2-DE and MS technology have been performed to compare different fiber elongation and/or secondary cell wall synthesis stages in WT cotton [25], and to compare early fiber elongation between WT and a lintless mutant [26]. For example, using a proteomic approach, an ascorbate peroxidase (APX) enzyme was found to accumulate significantly during the fast fiber cell elongation period [27].
The fuzzless–lintless cotton mutant (fl) produces mutant glabrous ovules that are ideal for comparison with WT fiber-bearing ovules in order to obtain fiber-enriched transcriptome or proteome profiles. Using the fl mutant, researchers have characterized some of the molecular mechanisms and several genes important for fiber initiation and elongation [13], [20], [22], [24]. Understanding the exact nature of this mutation would be a crucial step toward elucidating the genetic factors that regulate profiber cell differentiation and initiation. To address this issue, we performed a comparative proteomic investigation of proteins that are regulated in the immature ovules of the fl mutant [28], with the aim of identifying a set of proteins involved in the expression of the hairless phenotype.
Using 2-DE coupled with MALDI-TOF/TOF identification, we found 46 proteins in − 3 and 0 DPA ovules that were differentially expressed between fl and its original WT parent line (Xuzhou142). Most of the proteins (72%) were expressed at lower levels in the fl mutant than in the WT. The most common functional groups of the differentially expressed proteins were ROS-related and stress-responsive proteins, and carbohydrate metabolizing enzymes. The present results suggest some of the regulatory and functional pathways that differ in the fl mutant and provide information about the signal and/or metabolic networks that accompany fiber differentiation and initiation during early seed development.
Section snippets
Plant materials and sampling
The spontaneous fl mutant and WT parent line of an Upland cotton (G. hirsutum L. cv. Xuzhou 142) were grown in a greenhouse. For self-pollination, petals were tied with thread and tagged at − 1 DPA. Bolls were collected between 16:00–18:00 h at 0 DPA. There is commonly a 3-day flowering interval between the same node sites of adjacent fruit branches. This observation was used to identify buds at − 3 and − 2 DPA. The ovules were separated into aliquots for proteomic, metabolic, and quantitative
Fiber differentiation and initiation in WT versus fl cotton plants
It is generally believed that only some ovule epidermal cells develop into fibers. Although the distinct molecular identity of primordial fiber cells may be formed as early at − 3 DPA, cellular differences between fiber and non-fiber epidermal cells cannot be observed by TEM until − 1 DPA. The difficulty in isolating prefiber from non-fiber cells has hindered investigations of the molecular mechanisms involved in fiber differentiation and initiation. However, because the differences between the
Concluding remarks
To understand the molecular mechanisms governing cotton fiber differentiation and initiation, the proteomes of the fuzzless–lintless mutant and its parental wild-type line were compared. Overall, 46 proteins showed significant differences in expression between fl and WT ovules at − 3 or 0 DPA. The proteomic results were further validated by a ROS assay, metabolomic analysis, and qPCR analysis. The fl mutant proteome was consistent with increased ROS generation coupled with a decreased stress
Acknowledgment
This work has been supported by China Scientific and Technological Project of Transgenic New Biological Cultivar Breeding (2009ZX08009-113B), High Tech Project (2008AA10Z101), and the Program for 111 project in Ministry of Education, China (B08025). We are thankful to Dr Dayong Zhang, Institute of Genetics, Jiangsu Academy of Agricultural Sciences, for his help in SEM.
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