YUCCA genes are expressed in response to leaf adaxial-abaxial juxtaposition and are required for leaf margin development

During leaf development, the formation of leaf adaxial-abaxial polarity at the primordium stage is crucial for subsequent leaf expansion. However, little is known about the genetic control from polarity establishment to blade outgrowth. The leaf margin, comprising elongated margin cells and hydathodes, is thought to affect leaf expansion. Here, we show that mutants with defective leaf polarity or with loss of function in the multiple auxin-biosynthetic YUCCA (YUC) genes exhibited a similar abnormal leaf margin and less-expanded leaves. Leaf margins of these mutants contained fewer hydathodes and an increased number of cell patches in which the patterns of epidermal cells resembled those of hydathodes. The previously characterized leaf-abaxialized asymmetric leaves2 (as2) revoluta (rev) and leaf-adaxialized kanadi1 (kan1) kan2 double mutants both produce finger-shaped, hydathode-like protrusions on adaxial and abaxial leaf surfaces, respectively. YUCs are required for formation of the protrusions, as those produced by as2 rev and kan1 kan2 were absent in the yuc1 yuc2 yuc4 triple mutant background. Expressions of YUC1, YUC2, and YUC4 were spatially regulated in the leaf, being associated with hydathodes in wild-type leaves and protrusions on as2 rev and kan1 kan2 leaves. In addition, inhibition of auxin transport by treatment of seedlings with N-(1-naphtyl) phtalamic acid or disruption of the auxin gradient by transforming plants with the 35S:YUC1 construct also blocked leaf margin development. Collectively, our data show that expressions of YUCs in the leaf respond to the adaxial-abaxial juxtaposition, and that the activities of auxin mediate leaf margin development, which subsequently promotes blade outgrowth.     

The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22alpha-hydroxylation steps in brassinosteroid biosynthesis.

dwarf4 (dwf4) mutants of Arabidopsis display a dwarfed phenotype due to a lack of cell elongation. Dwarfism could be rescued by the application of brassinolide, suggesting that DWF4 plays a role in brassinosteroid (BR) biosynthesis. The DWF4 locus is defined by four mutant alleles. One of these is the result of a T-DNA insertion. Plant DNA flanking the insertion site was cloned and used as a probe to isolate the entire DWF4 gene. Sequence analysis revealed that DWF4 encodes a cytochrome P450 monooxygenase with 43% identity to the putative Arabidopsis steroid hydroxylating enzyme CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM. Sequence analysis of two other mutant alleles revealed deletions or a premature stop codon, confirming that DWF4 had been cloned. This sequence similarity suggests that DWF4 functions in specific hydroxylation steps during BR biosynthesis. In fact, feeding studies utilizing BR intermediates showed that only 22alpha-hydroxylated BRs rescued the dwf4 phenotype, confirming that DWF4 acts as a 22alpha-hydroxylase.     

Selective interaction of triazole derivatives with DWF4, a cytochrome P450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in planta

Brassinazole, a synthetic chemical developed in our laboratory, is a triazole-type brassinosteroid biosynthesis inhibitor that induces dwarfism in various plant species. The target sites of brassinazole were investigated by chemical analyses of endogenous brassinosteroids (BRs) in brassinazole-treated Catharanthus roseus cells. The levels of castasterone and brassinolide in brassinazole-treated plant cells were less than 6% of the levels in untreated cells. In contrast, campestanol and 6-oxocampestanol levels were increased, and levels of BR intermediates with hydroxy groups on the side chains were reduced, suggesting that brassinazole treatment reduced BR levels by inhibiting the hydroxylation of the C-22 position. DWF4, which is an Arabidopsis thaliana cytochrome P450 isolated as a putative steroid 22-hydroxylase, was expressed in Escherichia coli, and the binding affinity of brassinazole and its derivatives to the recombinant DWF4 were analyzed. Among several triazole derivatives, brassinazole had both the highest binding affinity to DWF4 and the highest growth inhibitory activity. The binding affinity and the activity for inhibiting hypocotyl growth were well correlated among the derivatives. In brassinazole-treated A. thaliana, the CPD gene involved in BR biosynthesis was induced within 3 h, most likely because of feedback activation caused by the reduced levels of active BRs. These results indicate that brassinazole inhibits the hydroxylation of the C-22 position of the side chain in BRs by direct binding to DWF4 and that DWF4 catalyzes this hydroxylation reaction.     

Shaping up: the genetic control of leaf shape

Leaf initiation at the shoot apical meristem involves a balance between cell proliferation and commitment to make primordia. Several genes, such as CLAVATA1, CLAVATA3, WUSCHEL, KNOTTED1, and PHANTASTICA, play key roles in these processes. When expressed in the leaf primordium, however, these 'meristem' genes can profoundly affect leaf shape and size, possibly by regulating hormone gradients and transport. The KNOTTED1-like genes are involved in regulating changes in hormonal levels. Recent studies have elaborated on the role that hormones, such as auxin, play in releasing biophysical constraints on leaf initiation and growth. Final leaf form is elaborated by a coordination of these hormonally regulated processes, cell division and cellular differentiation.     

Expression and functional analysis of <Emphasis Type="Italic">ZmDWF4</Emphasis>, an ortholog of <Emphasis Type="Italic">Arabidopsis DWF4</Emphasis> from maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

DWF4 encodes a rate-limiting mono-oxygenase that mediates 22α-hydroxylation reactions in the BR biosynthetic pathway and it is the target gene in the BR feedback loop. Knockout of DWF4 results in a dwarfed phenotype and other severe defects in Arabidopsis. Here we report on the isolation of the ZmDWF4 gene in maize. Sequence analysis revealed that the open reading frame of ZmDWF4 was 1,518 bp, which encodes a protein composed of 505 amino acid residues with a calculated molecular mass of 57.6 kD and a predicated isoelectric point (pI) of 9.54. Phylogenetic analysis indicated that ZmDWF4 was very close to the Arabidopsis DWF4. In young maize seedlings, the expression of ZmDWF4 in shoots was much higher than that in roots. The highest expression of ZmDWF4 was observed in husk leaves and the lowest in silks during flowering stage. The expression of ZmDWF4 in maize was significantly down regulated by exogenous brassinolide. A heterogeneous complementary experiment demonstrated that the defects of three Arabidopsis DWF4 mutants could be rescued by constitutive expression of ZmDWF4, with leaf expandability, inflorescence stem heights and fertile capabilities all restored to normal levels. Increases in seed and branch number as well as the height of florescence stem were observed in the over-expressed transformants. These findings suggest that ZmDWF4 may be an ortholog gene of Arabidopsis DWF4 and responsible for BR biosynthesis in maize. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Brassinosteroids control the proliferation of leaf cells of Arabidopsis thaliana

The growth of leaves in the model plant, Arabidopsis thaliana (L.) Heynh., is determined by the extent of expansion of individual cells and by cell proliferation. Mutants of A. thaliana with known defects in the biosynthesis or perception of brassinosteroids develop small leaves. When the leaves of brassinosteroid-related mutants, det2 (de-etiolated2 = cro1) and dwf1 (dwarf1 = cro2) were compared to wild-type plants, an earlier cessation of leaf expansion was observed; a detailed anatomical analysis further revealed that the mutants had fewer cells per leaf blade. Treatment of the det2 mutants with the brassinosteroid, brassinolide, reversed the mutation and restored the potential for growth to that of the wild type. Restoration of leaf size could not be explained solely on the basis of an increase in individual cell volume, thus suggesting that brassinosteroids play a dual role in regulating cell expansion and proliferation.     

Geographic variation and plasticity of leaf shape and size in Begonia dregei and B. homonyma (Begoniaceae)

The distinction between Begonia dregei and B. homonyma has hitherto been based on differences in the shape and size of leaves. A large sample of field-collected leaves has been examined using quantitative methods to test the validity of this distinction. Comparisons of leaf shape, with and without the inclusion of size-related variables, revealed three groups with a small amount of overlap, two of which correspond to B. dregei and one to B. homonyma. Effects of environmental conditions on leaf shape and size were examined in plants from four populations grown at two light intensities and compared with field collections. Leaf size varied little with environment, while margin roughness varied with the different conditions and there was variation among populations in the magnitude and direction of responses. These results show that leaf shape and size can be used as valid discriminators of groups within this taxon. Resolution of the taxonomy of this group will require consideration of heterogeneity within each of the three groups based on leaf shape and size.     

DWARF4 accumulation in root tips is enhanced via blue light perception by cryptochromes

Brassinosteroid (BR) signalling is known to be coordinated with light signalling in above ground tissue. Many studies focusing on the shade avoidance response in above ground tissue or hypocotyl elongation in darkness have revealed the contribution of the BR signalling pathway to these processes. We previously analysed the expression of DWARF 4 (DWF4), a key BR biosynthesis enzyme, and revealed that light perception in above ground tissues triggered DWF4 accumulation in root tips. To determine the required wavelength of light and photoreceptors responsible for this regulation, we studied DWF4‐GUS marker plants grown in several monochromatic light conditions. We revealed that monochromatic blue LED light could induce DWF4 accumulation in primary root tips and root growth as much as white light, whereas monochromatic red LED could not. Consistent with this, a cryptochrome1/2 double mutant showed retarded root growth under white light whereas a phytochromeA/B double mutant did not. Taken together, our data strongly indicated that blue light signalling was important for DWF4 accumulation in root tips and root growth. Furthermore, DWF4 accumulation patterns in primary root tips were not altered by auxin or sugar treatment. Therefore, we hypothesize that blue light signalling from the shoot tissue is different from auxin and sugar signalling.     

The Arabidopsis DIMINUTO/DWARF1 gene encodes a protein involved in steroid synthesis.

We have identified the function of the Arabidopsis DIMINUTO/DWARF1 (DIM/DWF1) gene by analyzing the dim mutant, a severe dwarf with greatly reduced fertility. Both the mutant phenotype and gene expression could be rescued by the addition of exogenous brassinolide. Analysis of endogenous sterols demonstrated that dim accumulates 24-methylenecholesterol but is deficient in campesterol, an early precursor of brassinolide. In addition, we show that dim is deficient in brassinosteroids as well. Feeding experiments using deuterium-labeled 24-methylenecholesterol and 24-methyldesmosterol confirmed that DIM/DWF1 is involved in both the isomerization and reduction of the Delta24(28) bond. This conversion is not required in cholesterol biosynthesis in animals but is a key step in the biosynthesis of plant sterols. Transient expression of a green fluorescent protein-DIM/DWF1 fusion protein and biochemical experiments showed that DIM/DWF1 is an integral membrane protein that most probably is associated with the endoplasmic reticulum.     

Organ shape and size: a lesson from studies of leaf morphogenesis

Control of the shape and size of indeterminate organs, such as roots and stems, is directly related to the control of the shape and size of the cells in these organs, as predicted by orthodox cell theory. For example, the polarity-dependent growth of leaf cells directly affects the polar expansion of leaves. Thus, the control of leaf shape is related to the control of the shape of cells within the leaf, as suggested by cell theory. By contrast, in determinate organs, such as leaves, the number of cells does not necessarily reflect organ shape or size. Genetic evidence shows that a compensatory system(s) is involved in leaf morphogenesis, and that an increase in cell volume can be triggered by a decrease in cell number and vice versa. Studies of chimeric leaves also suggest interaction between leaf cells that coordinates the behaviour of these cells at the organ level. Moreover, leaf size also appears to be coordinated at the whole-plant level. The recently hypothesised neo cell theory describes how leaf shape- and size-control mechanisms control leaf shape at the organ-level via cell-cell interaction.     

Spatiotemporal variation in leaf size and shape in response to climate

叶片的形态变异是研究植物响应气候变化的重要指标之一。叶片大小与形状影响着植物与外界环境的碳、水等物质交换和能量交换。然而,叶片大小与形状如何响应气候变化仍缺乏研究。此外,大多研究忽略了叶大小与形状在时间和空间上的种内变异,而只关注种间变异。我们收集了中国98年(1910-2008年)7个代表性双子叶植物的植物标本馆标本6000多份,并测量了它们叶片的叶长和宽度。我们探索了叶片大小(叶长、叶宽和长×宽乘积)的地理模式和时间趋势,(即研究了降水量和温度随时间和空间的变化对叶片大小和形状变化的影响。在控制了标本采集时间的影响后,全部物种的平均叶大小沿着纬度梯度的增加而减小,但这一关系在不同物种间存在差异。叶大小和形状的空间变异与温度和降水正相关。当控制了采集地点的影响后,全部物种的平均叶大小随着时间有变大的趋势。大多数物种叶大小的时间变化与降水正相关,而叶形状的时间变化则与温度正相关。我们的研究指出叶大小和形状在时空上的种内变异显著受气候的影响,同时说明植物标本可以为研究植物形态对气候变化的响应提供素材。     

植物叶发育调控机理研究的进展

在植物的营养生长阶段,叶原基从植物地上部分顶端分生组织的周边区形成,在一系列细胞分裂和分化程序的指导下,最终发育成叶。近年来,通过遗传学和分子生物学研究已经鉴定和克隆了一批参与叶发育调控的关键基因,植物激素在叶原基的诱导和叶形态建成中也起十分重要的作用。目前这个领域的主要研究工作是鉴定调控叶发育的新基因并且解释叶调控基因之间的相互作用,同时了解基因调控和植物激素作用之间的关系。     

植物叶发育调控机理研究的进展

在植物的营养生长阶段,叶原基从植物地上部分顶端分生组织的周边区形成,在一系列细胞分裂和分化程序的指导下,最终发育成叶。近年来,通过遗传学和分子生物学研究已经鉴定和克隆了一批参与叶发育调控的关键基因,植物激素在叶原基的诱导和叶形态建成中也起十分重要的作用。目前这个领域的主要研究工作是鉴定调控叶发育的新基因并且解释叶调控基因之间的相互作用,同时了解基因调控和植物激素作用之间的关系。