The last reaction producing brassinolide is catalyzed by cytochrome P-450s, CYP85A3 in tomato and CYP85A2 in Arabidopsis

Brassinosteroids are steroidal hormones essential for the growth and development of plants. Brassinolide, the most biologically active brassinosteroid, has a seven-membered lactone ring that is formed by a Baeyer-Villiger oxidation of its immediate precursor castasterone. Despite its potential key role in controlling plant development, brassinolide synthase has not been identified. Previous work has shown that the formation of castasterone from 6-deoxocastasterone is catalyzed by members of the CYP85A family of cytochrome P-450 monooxygenases. A null mutation in the tomato Dwarf (CYP85A1) gene, extreme dwarf (d(x)), causes severe dwarfism due to brassinosteroid deficiency, but the d(x) mutant still produces fruits. Here, we show that d(x) fruits contain brassinolide at a higher level than wild-type fruits and that a new CYP85A gene, CYP85A3, is preferentially expressed in tomato fruits. Tomato CYP85A3 catalyzed the Baeyer-Villiger oxidation to produce brassinolide from castasterone in yeast, in addition to the conversion of 6-deoxocastasterone to castasterone. We also show that Arabidopsis CYP85A2, which was initially characterized as castasterone synthase, also has brassinolide synthase activity. Exogenous application of castasterone and brassinolide to the Arabidopsis cyp85a1/cyp85a2 double mutant suggests that castasterone can function as an active brassinosteroid but that its conversion into brassinolide is necessary for normal vegetative development in Arabidopsis. We postulate that castasterone is the major active brassinosteroid during vegetative growth in tomato, whereas brassinolide may play an organ-specific role in fruit development in this species.     

Genetic Analysis of Growth-Regulator-Induced Parthenocarpy in Arabidopsis

In Arabidopsis, seedless silique development or parthenocarpy can be induced by the application of various plant growth regulators (PGRs) to unfertilized pistils. Ecotype-specific responses were observed in the Arabidopsis ecotypes Columbia and Landsberg relative to the type of PGR and level applied. The parthenocarpic response was greatest in ecotype Landsberg, and comparisons of fruit growth and morphology were studied primarily in this ecotype. Gibberellic acid application (10 micromol pistil(-1)) caused development similar to that in pollinated pistils, while benzyladenine (1 micromol pistil(-1)) and naphthylacetic acid (10 micromol pistil(-1)) treatment produced shorter siliques. Naphthylacetic acid primarily modified mesocarp cell expansion. Arabidopsis mutants were employed to examine potential dependencies on gibberellin biosynthesis (ga1-3, ga4-1, and ga5-1) and perception (spy-4 and gai) during parthenocarpic silique development. Emasculated spy-4 pistils were neither obviously parthenocarpic nor deficient in PGR perception. By contrast, emasculated gai mutants did not produce parthenocarpic siliques following gibberellic acid application, but silique development occurred following pollination or application of auxin and cytokinin. Pollinated gai siliques had decreased cell numbers and morphologically resembled auxin-induced parthenocarpic siliques. This shows that a number of independent and possibly redundant pathways can direct hormone-induced parthenocarpy, and that endogenous gibberellins play a role in regulating cell expansion and promoting cell division in carpels.     

Maternal control of seed size by EOD3/CYP78A6 in Arabidopsis thaliana

Seed size in higher plants is coordinately determined by the growth of the embryo, endosperm and maternal tissue, but relatively little is known about the genetic and molecular mechanisms that set final seed size. We have previously demonstrated that Arabidopsis DA1 acts maternally to control seed size, with the da1-1 mutant producing larger seeds than the wild type. Through an activation tagging screen for modifiers of da1-1, we have identified an enhancer of da1-1 (eod3-1D) in seed size. EOD3 encodes the Arabidopsis cytochrome P450/CYP78A6 and is expressed in most plant organs. Overexpression of EOD3 dramatically increases the seed size of wild-type plants, whereas eod3-ko loss-of-function mutants form small seeds. The disruption of CYP78A9, the most closely related family member, synergistically enhances the seed size phenotype of eod3-ko mutants, indicating that EOD3 functions redundantly with CYP78A9 to affect seed growth. Reciprocal cross experiments show that EOD3 acts maternally to promote seed growth. eod3-ko cyp78a9-ko double mutants have smaller cells in the maternal integuments of developing seeds, whereas eod3-1D forms more and larger cells in the integuments. Genetic analyses suggest that EOD3 functions independently of maternal factors DA1 and TTG2 to influence seed growth. Collectively, our findings identify EOD3 as a factor of seed size control, and give insight into how plants control their seed size.     

A Mutation in the Arabidopsis TFL1 Gene Affects Inflorescence Meristem Development

We present the initial phenotypic characterization of an Arabidopsis mutation, terminal flower 1-1 (tfl1-1), that identifies a new genetic locus, TFL1. The tfl1-1 mutation causes early flowering and limits the development of the normally indeterminate inflorescence by promoting the formation of a terminal floral meristem. Inflorescence development in mutant plants often terminates with a compound floral structure consisting of the terminal flower and one or two subtending lateral flowers. The distal-most flowers frequently contain chimeric floral organs. Light microscopic examination shows no structural aberrations in the vegetative meristem or in the inflorescence meristem before the formation of floral buttresses. The wild-type appearance of lateral flowers and observations of double mutant combinations of tfl1-1 with the floral morphogenesis mutations apetala 1-1 (ap1-1), ap2-1, and agamous (ag) suggest that the tfl1-1 mutation does not affect normal floral meristems. Secondary flower formation usually associated with the ap1-1 mutation is suppressed in the terminal flower, but not in the lateral flowers, of tfl1-1 ap1-1 double mutants. Our results suggest that tfl1-1 perturbs the establishment and maintenance of the inflorescence meristem. The mutation lies on the top arm of chromosome 5 approximately 2.8 centimorgans from the restriction fragment length polymorphism marker 217.     

Characterization of a selenate-resistant Arabidopsis mutant. Root growth as a potential target for selenate toxicity

Screening an Arabidopsis (Arabidopsis thaliana) T-DNA mutant library for selenate resistance enabled us to isolate a selenate-resistant mutant line (sel1-11). Molecular and genetic characterization showed that the mutant contained a lesion in the SULTR1;2 gene that encodes a high affinity root sulfate transporter. We showed that SULTR1;2 is the only gene among 13 mutated genes of the Arabidopsis sulfate transporter family whose mutation conferred selenate resistance to Arabidopsis. The selenate resistance phenotype of the sel1-11 mutant was mirrored by an 8-fold increase of root growth in the presence of selenate as shown by the calculated lethal concentration values. The impairment of SULTR1;2 activity in sel1-11 resulted in a reduced (35)S-sulfate uptake capacity by both roots and calli and a reduced sulfate and selenate content in root, shoot, and calli. Comparing sulfate-to-selenate ratios instead of absolute sulfate and selenate contents in roots and shoots enabled us to gain better insight into the mechanism of selenate toxicity in Arabidopsis. Roots of the sel1-11 mutant line showed a higher sulfate to selenate ratio than that of wild-type roots, while there were no significant differences in sulfate to selenate ratios in shoots of wild-type and mutant lines. These results indicated that the mechanism that confers the selenate resistance phenotype to the sel1-11 line takes place rather in the roots. It might be in part the result of a lower selenate uptake and of a protective effect of sulfate against the toxic effects of selenate on root growth. These results revealed in plants a central and specific role of the transporter SULTR1;2 in selenate sensitivity; they further suggested that root growth and potentially the root tip activity might be a specific target of selenate toxicity in Arabidopsis.     

Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1

Three Arabidopsis cDNAs, MAM1, CYP79F1, and CYP83A1, required for aliphatic glucosinolate biosynthesis were introduced into Chinese cabbage by Agrobacterium tumefaciens-mediated transformation. The transgenic lines overexpressing MAM1 or CYP83A1 showed wild-type phenotypes. However, all the lines overexpressing CYP79F1 displayed phenotypes different from wild type with respect to the stem thickness as well as leaf width and shape. Glucosinolate contents of the transgenic plants were compared with those of wild type. In the MAM1 line M1-1, accumulation of aliphatic glucosinolates gluconapin and glucobrassicanapin significantly increased. In the CYP83A1 line A1-1, all the aliphatic glucosinolate levels were increased, and the levels of gluconapin and glucobrassicanapin were elevated by 4.5 and 2 fold, respectively. The three CYP79F1 transgenic lines exhibited dissimilar glucosinolate profiles. The F1-1 line accumulated higher levels of gluconapoleiferin, glucobrassicin, and 4-methoxy glucobrassicin. However, F1-2 and F1-3 lines demonstrated a decrease in the levels of gluconapin and glucobrassicanapin and an increased level of 4-hydroxy glucobrassicin.     

CYP90A1/CPD, a Brassinosteroid Biosynthetic Cytochrome P450 of Arabidopsis, Catalyzes C-3 Oxidation

Brassinosteroids (BRs) are steroidal phytohormones that regulate plant growth and development. Whereas in Arabidopsis the network-like routes of BR biosynthesis have been elucidated in considerable detail, the roles of some of the biosynthetic enzymes and their participation in the different subpathways remained to be clarified. We investigated the function of the cytochrome P450 monooxygenase CYP90A1/CPD, which earlier had been proposed to act as a BR C-23 hydroxylase. Our GC-MS and genetic analyses demonstrated that the cpd mutation arrests BR synthesis upstream of the DET2-mediated 5α reduction step and that overexpression of the C-23 hydroxylase CYP90C1 does not alleviate BR deficiency in the cpd mutant. In line with these results, we found that CYP90A1/CPD heterologously expressed in a baculovirus-insect cell system catalyzes C-3 oxidation of the early BR intermediates (22S)-22-hydroxycampesterol and (22R,23R)-22,23-dihydroxycampesterol, as well as of 6-deoxocathasterone and 6-deoxoteasterone. Enzyme kinetic data of CYP90A1/CPD and DET2, together with those of the earlier studied CYP90B1, CYP90C1, and CYP90D1, suggest that BR biosynthesis proceeds mainly via the campestanol-independent pathway.     

Isolation of an Arabidopsis thaliana Mutant,mto1, That Overaccumulates Soluble Methionine (Temporal and Spatial Patterns of Soluble Methionine Accumulation)

We isolated Arabidopsis thaliana mutants that are resistant to ethionine, a toxic analog of methionine (Met). One of the mutants was analyzed further, and it accumulated 10- to 40-fold more soluble Met than the wild type in the aerial parts during the vegetative growth period. When the mutant plants started to flower, however, the soluble Met content in the rosette region decreased to the wild-type level, whereas that in the inflorescence apex region and in immature fruits was 5- to 8-fold higher than the wild type. These results indicate that the concentration of soluble Met is temporally and spatially regulated and suggest that soluble Met is translocated to sink organs after the onset of reproductive growth. The causal mutation, designated mto1, was a single, nuclear, semidominant mutation and mapped to chromosome 3. Accumulation profiles of soluble amino acids suggested that the mutation affects a later step(s) in the Met biosynthesis pathway. Ethylene production of the mutants was only 40% higher than the wild-type plants, indicating that ethylene production is tightly regulated at a step after Met synthesis. This mutant will be useful in studying the translocation of amino acids, as well as regulation of Met biosynthesis and other metabolic pathways related to Met.     

Characterization of a Phosphate-Accumulator Mutant of Arabidopsis thaliana

We have characterized a novel mutation of Arabidopsis thaliana at a locus designated pho2. pho2 mutants accumulated up to 3-fold more total P in leaves, mostly as inorganic phosphate (Pi), than wild-type seedlings. In addition, we isolated a mutant (locus designated pho1-2, an allelle of pho1-1 described by Y. Poirier, S. Thoma, C. Somerville, J. Schiefelbein [1991] Plant Physiol 97: 1087-1093) with low Pi concentrations in leaves. When grown under high transpiration conditions, leaves of pho2 seedlings became severely P intoxicated, whereas shoots of pho1-2 mutants were P deficient and wild-type seedlings were normal. A pho1/pho2 double mutant resulting from a cross between the single mutants was identified in the F2 generation and shown to have a pho1 phenotype. Prior to the development of P toxicity symptoms, P was the only mineral nutrient whose concentration was greater in pho2 mutants than wild-type seedlings. Compared to wild-type, pho2 mutants had greater Pi concentrations in stems, siliques, and seeds, but roots of pho2 mutants had similar or lower Pi concentrations than either pho1 mutants or wild-type seedlings. We suggest that the pho2 mutation affects a function normally involved in regulating the concentration of Pi in shoots of Arabidopsis.     

AtMRD1 and AtMRU1, two novel genes with altered mRNA levels in the methionine over-accumulating mto1-1 mutant of Arabidopsis thaliana

The mto1-1 mutant of Arabidopsis thaliana over-accumulates soluble methionine (Met) up to 40-fold higher than that in its Col-0 wild type. In order to identify genes regulated by altered Met concentrations, microarray analysis of gene expression in young rosettes and developing siliques of the mto1-1 mutant were performed. Expression of selected genes was then examined in detail in three developmental stages of the mto1-1 mutant using a combination of Northern hybridisation analysis and real-time PCR. Eight genes were identified that had altered mRNA accumulation levels in the mto1-1 mutant compared to that in wild-type plants. Three of the genes have known roles in plant development unrelated to amino acid biosynthesis. One other gene up-regulated specifically in mto1-1 rosettes shared similarity with the embryo-specific protein 3 (ATS3). Two novel genes, referred to as AtMRD1 and AtMRU1, were also identified that were expressed in a developmental manner in wild-type Col-0 and do not share sequence similarity with genes of known function. AtMRD1 was strongly down-regulated in both rosette and young silique tissues of the mto1-1 mutant. AtMRU1 was up-regulated approximately 3-fold in young mto1-1 rosettes and exhibited a developmental response to the mto1-1 mutation.     

bor1-1, an Arabidopsis thaliana mutant that requires a high level of boron.

bor1-1 (high boron requiring), an Arabidopsis thaliana mutant that requires a high level of B, was isolated. When the B concentration in the medium was reduced to 3 microM, the expansion of rosette leaves was severely affected in bor1-1 but not in wild-type plants. In a medium containing 30 microM B the mutant grew normally but showed female sterility, whereas the wild type was able to set seeds. These defects of the bor1-1 mutant were not detected with supplementation of 100 microM B. In vivo concentrations of B in bor1-1 mutants were lower than those of the wild type, especially in the inflorescence stems. Tracer experiments using 10B suggested that the mutant has defects in uptake and/or translocation of B. The mutation was mapped on the lower arm of chromosome 2.     

Highly pleiotropic functions of CYP78As and AMP1 are regulated in non-cell-autonomous/organ-specific manners

The cytochrome P450 CYP78A5/KLUH in Arabidopsis thaliana is predicted to be involved in the synthesis of a mobile signal molecule that has a pleiotropic function that is distinct from classical phytohormones. CYP78A5 has five close relatives in Arabidopsis. We first investigated their functions, focusing on the plastochron, leaf size, and leaf senescence. Our analyses revealed that CYP78A5 and CYP78A7 are involved in the plastochron and leaf size, and CYP78A6 and CYP78A9 are involved in leaf senescence. Complementation analyses using heterologous promoters and expression analyses suggested that CYP78A isoforms have a common biochemical function and are functionally differentiated via organ-specific expression. The altered meristem program1 (amp1) carboxypeptidase mutant shows a phenotype very similar to that of the cyp78a5 mutant. Complementation analyses using boundary and organizing center-specific promoters suggested that both CYP78A5 and AMP1 act in a non-cell-autonomous manner. Analyses of multiple cyp78a mutants and crosses between cyp78a and amp1 mutants revealed that AMP1/LIKE AMP1 (LAMP1) and CYP78A isoforms regulate plastochron length and leaf senescence in the same genetic pathway, whereas leaf size is independently regulated. Furthermore, we detected feedback regulation between CYP78A6/CYP78A9 and AMP1 at the gene expression level. These observations raise the possibility that AMP1 and CYP78A isoforms are involved in the synthesis of the same mobile signal molecule, and suggest that AMP1 and CYP78A signaling pathways have a very close, albeit complex, functional relationship.

ALTERED MERISTEM PROGRAM1 and isoforms of the cytochrome P450 CYP78A regulate plastochron and leaf senescence in non-cell-autonomous/organ-specific manners, and have a close, albeit complex, and functional relationship.     

CYP78A5 encodes a cytochrome P450 that marks the shoot apical meristem boundary in Arabidopsis

The normal development of shoot structures depends on controlling the growth, proliferation and differentiation of cells derived from the shoot apical meristem. We have identified the CYP78A5 gene encoding a putative cytochrome P450 monooxygenase that is the first member of the CYP78 family from Arabidopsis. This gene is strongly expressed in the peripheral regions of the vegetative and reproductive shoot apical meristems, defining a boundary between the central meristematic zone and the developing organ primordia. In addition, CYP78A5 shows a dynamic pattern of expression during floral development. Overexpression of CYP78A5 affects multiple cell types, causing twisting and kinking of the stem and defects in floral development. To define the relationship of CYP78A5 to genes controlling meristem function, we examined CYP78A5 expression in plants mutant for SHOOT MERISTEMLESS, ZWILLE and ARGONAUTE, and have found that CYP78A5 expression is altered in these mutant backgrounds. We propose that CYP78A5 has a role in regulating directional growth in the peripheral region of the shoot apical meristem in response to cues established by genes regulating meristem function.     

A Seed Shape Mutant of Arabidopsis That Is Affected in Integument Development

A seed shape mutant of Arabidopsis was isolated from an ethyl methanesulfonate-treated population. Genetic analysis revealed that the heart-shaped phenotype was maternally inherited, showing that this is a testa mutant. This indicated the importance of the testa for the determination of the seed shape. This recessive aberrant testa shape (ats) gene was located at position 59.0 on chromosome 5. A comparison was made between ovules and developing and mature seeds of the wild type and of the mutant using light and scanning electron microscopy. We showed that the mutant seed shape is determined during the first few days after fertilization, when the embryo occupies only a very small part of the seed. The integuments of ats ovules consisted of only three rather than five cell layers. In double mutants, the effect of ats was additive to other testa mutations, such as transparent testa, glabra (ttg), glabrous2 (gl2), and apetala2 (ap2). The ats mutation resulted in a reduced dormancy, which was maternally inherited. This effect of a testa mutation on germination was also seen in ttg seeds, in which the outer layer of the testa was disturbed. This indicated the importance of the testa as a factor in determining dormancy in Arabidopsis.     

A mutation (Pro-30 to Leu) in CYP21 represents a potential nonclassic steroid 21-hydroxylase deficiency allele

The mild nonclassic form of steroid 21-hydroxylase deficiency is one of the most common autosomal recessive disorders in humans, occurring in almost 1% of caucasians and about 3% of Ashkenazi Jews. Many patients with this disorder carry a Val-281----Leu missense mutation in the CYP21 gene. This and most other mutations causing 21-hydroxylase deficiency are normally present in the CYP21P pseudogene and have presumably been transferred to CYP21 by gene conversion. To identify other potential nonclassic alleles, we used recombinant vaccinia virus to express two mutant enzymes carrying the mutations Pro-30----Leu (normally present in CYP21P) and Ser-268----Thr (considered a normal polymorphism of CYP21). Whereas the activity of the protein carrying the Ser----Thr mutation was indeed indistinguishable from the wild type, the enzyme with the Pro----Leu substitution had 60% of wild-type activity for 17-hydroxyprogesterone and about 30% of normal activity for progesterone when assayed in intact cells. When kinetic analysis of the latter mutant enzyme was performed in cellular lysates, the first order rate constants (maximum velocity/dissociation constant) for both substrates were reduced 10- to 20-fold compared with those for the wild-type enzyme. Pro-30 is conserved in many microsomal P450 enzymes and may be important for proper orientation of the enzyme with respect to the aminoterminal transmembrane segment. The Pro----Leu mutation was present in 5 of 18 patients with nonclassic 21-hydroxylase deficiency, suggesting that this mutation indeed acts as a nonclassic deficiency allele.     

细胞色素P450基因CYP78A5／KLUH控制拟南芥叶发育的时期转换

细胞色素P450蛋白CYP78A5／KLUH主要以非细胞自主性的方式调控了拟南芥器官大小的发育。我们对一个新的cyp78a5（sALK_024697）突变体的研究表明,CYP78A5基因还参与控制了拟南芥叶发育的时期转换。cyp78a5突变体中幼年态叶（juvenile leaf）向转换期叶（transition leaf）的发育时期推迟,而且没有成年态叶（adult leaf）形成。遗传分析表明CYP78A5基因可能与SUPPRESSOR OF GENE SILENCING3（SGS3）基因作用在同一个遗传调控途径控制叶片发育时期的转换。