Functional analysis of Gossypium hirsutum cellulose synthase catalytic subunit 4 promoter in transgenic Arabidopsis and cotton tissues
Research highlights
▶ Spatial regulation of cotton fiber GhCesA4 promoter was similar between cotton tissues and transgenic Arabidopsis. ▶ One upstream region (−2574/−1824) of the GhCesA4 promoter was involved in up-regulating GhCesA4 expression in trichomes and root vascular tissues. ▶ The −1824/−1355 region of the GhCesA4 promoter was involved in down-regulating GhCesA4 expression in vascular tissues. ▶ The −693/−320 region of the GhCesA4 promoter was necessary for basal expression of GhCesA4 in cotton roots as well as Arabidopsis trichomes and roots. ▶ GhCesA4 expression was differentially regulated in transgenic Arabidopsis by NAA, kinetin, and brassinosteroids.
Introduction
Cellulose, the most abundant biopolymer in nature, organizes into microfibrils in plant cell walls, providing strength and flexibility to plant tissues. Cellulose is synthesized by a plasma membrane associated, multisubunit enzyme called cellulose synthase [1]. The first plant cellulose synthase catalytic subunits (CesAs) were identified by comparing cotton fiber ESTs with bacterial cellulose synthase [2]. Extensive searches for CesA genes and mutant phenotypes in a model plant, Arabidopsis revealed that at least 10 different cellulose synthase catalytic subunits (AtCesAs) exist [3]. Three genes, AtCesA1, AtCesA3, and AtCesA6 are expressed during primary cell wall (PCW) biosynthesis in roots and hypocotyls [4], [5], [6]. Another set of three genes, AtCesA4, AtCesA7, and AtCesA8 are expressed during secondary cell wall (SCW) biosynthesis in Arabidopsis xylem cells [7], [8], [9]. GhCesA1 [2] and GhCesA4 [10] isolated from cotton fibers are orthologs of AtCesA8 [9] involved in SCW cellulose biosynthesis in Arabidopsis [11]. The sequence comparison of GhCesA1 (U58283) and GhCesA4 (AF413210) with two BACs containing homologous GhCesA1 genes showed that GhCesA1 and GhCesA4 are homologous genes of the D and A subgenomes of allotetraploid Gossypium hirsutum, respectively [12]. Northern blot analyses showed that GhCesA1, 2, and 4 are specifically expressed in fiber tissues [2], [10]. During cotton fiber development, transcript levels of GhCesA1, 2, and 4 are significantly up-regulated at the transition from PCW to SCW biosynthesis [2], [10].
Cotton (G. hirsutum L.) fibers are unicellular trichomes that differentiate from epidermal cells of developing cotton ovules [13]. Cotton fiber development is divided into four overlapping stages, (1) initiation, (2) PCW biosynthesis for fiber elongation, (3) SCW biosynthesis for cellulose production, and (4) maturation [14]. Fiber initiation starts a day before up to a day or two after anthesis, and the initials enter into the elongation phase immediately. During the PCW stage, a thin PCW is deposited in elongating fibers and cotton fibers elongate up to 3–6 cm for 2–3 weeks. The SCW stage initiates approximately 14–16 days post-anthesis (DPA), overlapping the final PCW stage. Mature fibers exhibit thickened SCW composed of nearly pure cellulose. At the transition from PCW to SCW biosynthesis in cotton fiber, synthesis of other cell wall polymers ceases and the rate of cellulose synthesis in cotton fibers are estimated to increase nearly 100-fold in vivo [15].
Although most genes involved in fiber elongation and cellulose biosynthesis in developing cotton fibers are transcriptionally regulated [2], [10], [11], [13], [14], [15], difficulties in regenerating transgenic cotton have impeded the study of transcriptional regulation of cotton fiber genes. To circumvent the lengthy and labor intensive tissue culture procedures for constructing multiple transgenic lines of cotton plants, most functional analyses of cotton promoters have been studied in transgenic tobacco or Arabidopsis [16], [17], [18], [19], [20], [21], [22]. Since cotton fibers are seed trichomes, numerous cotton fiber specific promoters were studied in leaf trichomes of heterologous transgenic plants despite the limited understanding of potential similarities for transcriptional regulation between seed trichomes and leaf trichomes. Analyses of cotton fiber specific promoters using heterologous transgenic plants led to the identification of MYB and L1 as promoter motifs for trichome specific expression [18], and an AT-rich motif for repressing gene expression in non-fiber tissues [20]. In spite of these advances, whether the developmental and transcriptional regulation of cotton genes can similarly occur in heterologous transgenic plants is unknown. Therefore, cotton fiber specific promoter motifs identified from heterologous transgenic plants must be further verified in cotton tissues.
Although spatial regulations of cotton promoters involved in PCW biosynthesis during fiber development have been extensively studied using transgenic tobacco or Arabidopsis [16], [17], [18], [19], [20], [21], comparatively less is known about cotton promoter activity involved in SCW biosynthesis during fiber development. A recent promoter activity assay of GhCesA4, a gene involved in SCW biosynthesis of developing cotton fibers, showed that GhCesA4 was preferentially expressed in vascular tissues and induced by a synthetic auxin, NAA when a GUS reporter regulated by a short version (−1407/+106) of the GhCesA4 promoter named P1482 was analyzed in transgenic tobacco [22].
To understand transcriptional regulation of SCW cellulose biosynthesis in cotton fibers, our group has also studied GhCesA4 promoter activity using a longer version (−2574/+56) of the GhCesA4 promoter (AF413210) isolated from G. hirsutum DPL90 [10], [13]. In our study, we evaluated GhCesA4 promoter activity by monitoring GUS expression in cotton tissues as well as transgenic Arabidopsis transformed stably or transiently regulated by the GhCesA4 promoter (−2574/+56) or progressively smaller promoters. Consistent with the results reported by Wu et al. [22], we found that GUS activity regulated by one of the progressively deleted GhCesA4 promoters (−1.355/+56), a size similar to P1482 (−1407/+106), was mainly detected in vascular tissues in both cotton tissues and transgenic Arabidopsis. Furthermore, we report here that one upstream region (−1.824/−1355) of the GhCesA4 promoter is involved in down-regulating GhCesA4 expression in vascular tissues and another upstream region (−2574/−1824) is involved in up-regulating GhCesA4 expression in trichomes and roots. For basal expression of GhCesA4 in both transgenic Arabidopsis and cotton roots, one downstream region (−693/−320) was required. We also show that several phytohormones differentially regulated GhCesA4 promoter activity in various tissues at different developmental stages of transgenic Arabidopsis. In contrast to the previous report [22], our study shows by using the longer version of GhCesA4 promoter (−2574/+56) that GhCesA4 promoter activity was down-regulated by NAA in transgenic Arabidopsis.
Section snippets
Plant materials and growth conditions
Cotton plants (G. hirsutum L. TM-1) were grown in the field at the USDA, ARS, Southern Regional Research Center, New Orleans. Developing bolls were collected by 9 am at 4-day intervals from 8 through 24 DPA and fibers were immediately harvested and frozen in liquid nitrogen. Fully grown leaves (15 cm in diameter), expanding young leaves (5 cm in diameter), hypocotyls and roots were harvested from 1 or 6-week-old plants grown in a greenhouse at 25–32 °C. All tissues were frozen in liquid nitrogen,
Sequence analysis of GhCesA4 promoter
The upstream sequences from the translational start codon of GhCesA4 consist of 2681 nucleotides (Fig. 1). Computational analysis [28] showed that the putative transcriptional start site of GhCesA4 is located 107 nucleotides upstream from the translational start codon and the transcriptional start site is marked as +1 (Fig. 1). By using two algorithms of PLACE [29] and PlantCARE [30], a number of putative promoter motifs were identified within the GhCesA4 promoter composed of 2574 nucleotides.
Discussion
In this study, we first analyzed GhCesA4 promoter activity quantitatively and histochemically with cauline leaves from transgenic Arabidopsis. Since GhCesA4 was developmentally regulated during leaf vascular development in transgenic Arabidopsis and each rosette leaf has different developmental stage according to the order of appearance from the leaf primordium, we used two similar sized (∼0.5 cm) cauline leaves at the same position from the stem for each transgenic line to perform both
Acknowledgements
This work was supported by the USDA-ARS, NASA, the National Science Foundation, and the Louisiana State Support Program of Cotton Incorporated. The authors gratefully acknowledge Dr. David M. Stelly, Dr. C. Wayne Smith, and the Department of Soil & Crop Sciences at Texas A&M University for their generosity and hospitality in hosting us after our laboratories in New Orleans were damaged from Hurricane Katrina. We also thank Drs. Jeffrey Cary and Sukumar Saha of USDA-ARS for critically reading
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