Arabidopsiscyp51Mutant Shows Postembryonic Seedling Lethality Associated with Lack of Membrane Integrity

Abstract

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14a-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14a-methyl-D 8 -sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14a-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.Sterols are ubiquitous among most eukaryotic organisms. Bulk sterols, such as cholesterol in animals, ergosterol in yeast (Saccharomyces cerevisiae), and sitosterol in plants, serve to regulate membrane fluidity and permeability and indirectly affect the activity and distribution of integral membrane proteins, including enzymes, ion channels, and signal transduction components (Hartmann, 1998). These sterols also serve as precursors for bioactive molecules, such as mammalian steroid hormones, plant brassinosteroid (BR) hormones, and insect ecdysteroids to control developmental processes (Clouse, 2000). BRs are plant hormones that have important roles in plant development, including cell elongation, division, vascular differentiation, senescence, and stress responses (Clouse and Sasse, 1998). Plant sterols are also substrates for the synthesis of a wide range of secondary metabolites (Hartmann, 1998). Recently, the implication of sitosterol glucoside in the synthesis of cellodextrins has been proposed (Peng et al., 2002), and a link between sterol biosynthesis and cellulose production has been further documented (Schrick et al., 2004a).Plant sterols derive from cycloartenol via a series of reactions, including methylation, reduction, isomerization, and desaturation. The molecular genetic and biochemical analyses using Arabidopsis (Arabidopsis thaliana) mutants for genes encoding sterol biosynthe...

Cytochrome P450CYP710AEncodes the Sterol C-22 Desaturase inArabidopsisand Tomato

“…The expression of Arabidopsis CYP710A genes was suggested to be under strict regulation in different tissues and organs (Figure 8), suggesting the possibility that phenotypic alterations may be observed in definite tissues at specific developmental stages. It has been reported that another P450 gene involved in sterol biosynthesis, CYP51G1, was strongly expressed in leaf vascular tissues (Kim et al, 2005), which was closely similar to the expression pattern of CYP710A1 involved in stigmasterol production (Figure 8). CYP51G1 (At1g11680) is the single functional gene (Kim et al, 2005) encoding obtusifoliol 14a-demetylase protein in Arabidopsis (Figure 1).…”

“…cvp1 plants displayed postembryonic vascular patterning defects (Carland et al, 2002), and SMT2 cosuppression lines contained higher levels of campesterol and lower levels of sitosterol, exhibiting developmental abnormalities such as reduced apical dominance and reduced fertility that cannot be restored by exogenous BRs (Schaeffer et al, 2001). Furthermore, it has recently been reported that a T-DNA insertion mutagenesis within the obtusifoliol 14a-demethylase gene CYP51G1 caused defects in membrane integrity and hypocotyl elongation, leading to postembryonic seedling lethality (Kim et al, 2005). These results indicate that altered sterol profiles due to the mutations both upstream and downstream of the SMT2 step affect membrane properties, thereby influencing signaling cascades involved in normal plant growth, such as cell polarity, auxin efflux, and ethylene signaling.…”

Sterol Methyl Oxidases Affect Embryo Development via Auxin-Associated Mechanisms

“…However, increasing evidence indicates that plant sterols per se can act as signaling molecules. Plant sterol biosynthetic mutants, such as smt1/cph, cpi1-1, cyp51A2, fackel (fk), and hyd1, could not be rescued by BR application (Diener et al, 2000;Jang et al, 2000;Schrick et al, 2000Schrick et al, , 2002Souter et al, 2002;Willemsen et al, 2003;Kim et al, 2005;Men et al, 2008). Sterols were found to have intimate interactions with plant hormones, such as auxin and ethylene.…”

Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms

“…As a result, the number of known CYP51 sequences exceeds 100. The reasons for the existence of homologous CYP51 genes in the same species or their precise functions remain unknown, though it was reported that only one of the two CYP51 genes from A. thaliana (CYP51A2) is functional, while the other is an expressed pseudogene [15]. Mammalian genomes contain only one CYP51 gene yet sometimes † Corresponding author: Michael R. Waterman, Tel.…”