Structure,organization and expression of the metallothionein gene family inArabidopsis

Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi. Recent results indicate that plants also possess functional metallothionein genes. Here we report the cloning and characterization of five metallothionein genes fromArabidopsis thaliana. The position of the single intron in each gene is conserved. The proteins encoded by these genes can be divided into two groups (MT1 and MT2) based on the presence or absence of a central domain separating two cysteine-rich domains. Four of the MT genes (MT1a,MT1c,MT2a andMT2b) are transcribed inArabidopsis. Several lines of evidence suggest that the fifth gene,MT1b, is inactive. There is differential regulation of the MT gene family. MT1 mRNA is expressed highly in roots, moderately in leaves and is barely detected in inflorescences and siliques. MT2a and MT2b mRNAs are more abundant in leaves, inflorescences and in roots from mature plants, but are also detected in roots of young plants, and in siliques. MT2a mRNA is strongly induced in seedlings by CUSO₄, whereas MT2b mRNA is relatively abundant in this tissue and levels increase only slightly upon exposure to copper.MT1a andMT1c are located within 2 kb of each other and have been mapped to chromosome 1.MT1b andMT2b map to separate loci on chromosome V, andMT2a is located on chromosome III. The locations of these MT genes are different from that ofCAD1, a gene involved in cadmium tolerance inArabidopsis.     

Structure, organization and expression of the metallothionein gene family inArabidopsis

Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi. Recent results indicate that plants also possess functional metallothionein genes. Here we report the cloning and characterization of five metallothionein genes fromArabidopsis thaliana. The position of the single intron in each gene is conserved. The proteins encoded by these genes can be divided into two groups (MT1 and MT2) based on the presence or absence of a central domain separating two cysteine-rich domains. Four of the MT genes (MT1a,MT1c,MT2a andMT2b) are transcribed inArabidopsis. Several lines of evidence suggest that the fifth gene,MT1b, is inactive. There is differential regulation of the MT gene family. MT1 mRNA is expressed highly in roots, moderately in leaves and is barely detected in inflorescences and siliques. MT2a and MT2b mRNAs are more abundant in leaves, inflorescences and in roots from mature plants, but are also detected in roots of young plants, and in siliques. MT2a mRNA is strongly induced in seedlings by CUSO₄, whereas MT2b mRNA is relatively abundant in this tissue and levels increase only slightly upon exposure to copper.MT1a andMT1c are located within 2 kb of each other and have been mapped to chromosome 1.MT1b andMT2b map to separate loci on chromosome V, andMT2a is located on chromosome III. The locations of these MT genes are different from that ofCAD1, a gene involved in cadmium tolerance inArabidopsis.     

Trichome expression of iaaM transgene influences their development and elongation in tobacco

The iaaM gene encodes a monooxygenase, an enzyme that can catalyze the synthesis of IAA from tryptophan and is functional in plants. We cloned the iaaM into the T-DNA region of a Ti binary vector pWM101 under the control of the cotton fiber specific E6 promoter, and the recombinant was transformed into Nicotiana tabacum WS38 via leaf disc infection with Agrobacterium tumefaciencs GV3101 that harbored the Ti plasmid. Some 25 transformed seedlings were screened out, and the trichomes of the transgenic leaves were both denser and lengthier. The E6 promoter-specific expression of iaaM in trichomes led to the more active auxin synthesis in the cells and allowed the transgenic leaves to develop tidier and longer trichomes. As more trichomes developed on the transgenic leaves, the leaf epidermis appeared fuzzier than the wild control. The research showed that iaaM expression in trichomes influenced the development of trichomes both during their initiation and elongation.     

Foliar trichome- and aquaporin-aided water uptake in a drought-resistant epiphyte Tillandsia ionantha Planchon

The atmospheric epiphyte Tillandsia ionantha is capable of surviving drought stress for 6 months or more without any exogenous water supply via an as of yet to be determined mechanism. When plants were soaked in water for 3 h, leaves absorbed a remarkably large amount of water (30–40% on the basis of fresh weight), exhibiting a bimodal absorption pattern. Radiolabeled water was taken up by the leaves by capillary action of the epidermal trichomes within 1 min (phase 1) and then transported intracellularly to leaf tissues over 3 h (phase 2). The removal of epidermal trichome wings from leaves as well as rinsing leaves with water significantly lowered the extracellular accumulation of water on leaf surfaces. The intracellular transport of water was inhibited by mercuric chloride, implicating the involvement of a water channel aquaporin in second-phase water absorption. Four cDNA clones (TiPIP1a, TiPIP1b, TiPIP1c, and TiPIP2a) homologous to PIP family aquaporins were isolated from the leaves, and RT-PCR showed that soaking plants in water stimulated the expression of TiPIP2a mRNA, suggesting the reinforcement in ability to rapidly absorb a large amount of water. The expression of TiPIP2a complementary RNA in Xenopus oocytes enhanced permeability, and treatment with inhibitors suggested that the water channel activity of TiPIP2a protein was regulated by phosphorylation. Thus, the high water uptake capability of T. ionantha leaves surviving drought is attributable to a bimodal trichome- and aquaporin-aided water uptake system based on rapid physical collection of water and subsequent, sustained chemical absorption.     

拟南芥2-甲基-6-叶绿基-1,4-苯醌甲基转移酶启动子的分离及表达特性分析

维生素E是一类人体必需的脂溶性抗氧化剂, 具有重要的生理功能。2-甲基-6-叶绿基-1,4-苯醌甲基转移酶(MPBQ MT)是天然维生素E合成途径中的关键酶之一, 催化MPBQ甲基化, 生成DMPBQ。从拟南芥分离了MPBQ MT基因1018bp的启动子序列, 构建了含该启动子和GUS报告基因的植物表达载体, 通过农杆菌介导转化拟南芥, 获得了转基因植株。GUS组织化学染色结果表明, 在MPBQ MT启动子驱动下, 报告基因GUS在拟南芥的茎、叶、花萼、雄蕊、种荚均有表达, 且在茎、叶、种荚中表达量较高, 而在根、花瓣和种子中则没有观察到GUS基因的表达, 表明MPBQ MT基因可能仅在拟南芥幼嫩茎、叶、种荚等绿色组织中特异性高表达。     

Photosynthesis in leaves and siliques of winter oilseed rape (Brassica napus L.)

The rate of photosynthesis and its relation to tissue nitrogen content was studied in leaves and siliques of winter oilseed rape (Brassica napus L.) growing under field conditions including three rates of nitrogen application (0, 100 or 200 kg N ha^-1) and two levels of irrigation (rainfed or irrigated at a deficit of 20 mm). The predominant effect of increasing N application under conditions without water deficiency was enhanced expansion of photosynthetically active leaf and silique surfaces, while the rate of photosynthesis per unit leaf or silique surface area was similar in the different N treatments. Thus, oilseed rape did not increase N investment in leaf area expansion before a decline in photosynthetic rate per unit leaf area due to N deficiency could be avoided. Much less photosynthetically active radiation penetrated into high-N canopies than into low-N canopies. The specific leaf area increased markedly in low light conditions, causing leaves in shade to be less dense than leaves exposed to ample light. In both leaves and siliques the photosynthetic rate per unit surface area responded linearly to increasing N content up to about 2 g m^-2, thus showing a constant rate of net CO₂ assimilation per unit increment in N (constant photosynthetic N use efficiency). At higher tissue N contents, photosynthetic rate responded less to changes in N status. Expressed per unit N, light saturated photosynthetic rate was three times higher in leaves than in silique valves, indicating a more efficient photosynthetic N utilization in leaves than in siliques. Nevertheless, from about two weeks after completion of flowering and onwards total net CO₂ fixation in silique valves exceeded that in leaves because siliques received much higher radiation intensities than leaves and because the leaf area declined rapidly during the reproductive phase of growth. Water deficiency in late vegetative and early reproductive growth stages reduced the photosynthetic rate in leaves and, in particular, siliques of medium- and high-N plants, but not of low-N plants.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

<Emphasis Type="Italic">Virola sebifera</Emphasis> Aubl. (Myristicaceae) leaf chemical composition and implications on leaf-cutter ant foraging choice

Leaf-cutter ant plant material choice is essential for colony maintenance and growth. Plant material is used as a substrate for cultivating symbiotic fungus, and the ants’ preference for particular leaves, tends to be determined by vegetal age-related physicochemical factors. The plant species Virola sebifera Aubl. (Myristicaceae), for example, shows a large number of leaf surface trichomes. Although non-glandular, V. sebifera trichomes may gradually retain an increasing amount of chemical compounds over the lifetime of the leaf. Thus, the present study aims to investigate the role of plant chemical compounds on Atta sexdens rubropilosa preference for V. sebifera leaves of different ages. For this purpose, the chemical composition of trichomes on young and senescent leaves was analyzed, and ants’ preference tested. The chemical compositions differ between V. sebifera young and senescent leaves, with triacontane (C30) predominance in young leaves and tetratriacontane (C34) predominance in senescent leaves. Ants’ preference choice was tested by randomly offering leaves of different ages to A. sexdens rubropilosa workers, following six different treatments: (1) young leaf fragments; (2) young leaf fragments with few trichomes removed; (3) loose trichomes from young leaves; (4) senescent leaf fragments; (5) senescent leaf fragments with few trichomes removed, and (6) loose trichomes from senescent leaves. Ants’ preference was observed for young leaves fragments with a few trichomes removed and also for young leaves loose trichomes. Ants’ preference might be due to specific volatile compounds (GLV) preset in V. sebifera young leaves. Results suggest occurrence of ants’ selectivity resulting from changes on trichomes chemical composition between V. sebifera leaves different age stages.     

The MIXTA-like Transcription factor MYB16 is a major regulator of cuticle formation in vegetative organs

Cuticle secreted on the surface of the epidermis of aerial organs protects plants from the external environment. We recently found that Arabidopsis MIXTA-like R2R3-MYB family members MYB16 and MYB106 regulate cuticle formation in reproductive organs and trichomes. However, the artificial miRNA (amiRNA)-mediated knockdown plants showed no clear phenotypic abnormality in vegetative tissues. In this study, we used RNA interference (RNAi) targeting MYB16 to produce plants with reduced expression of both MYB16 and MYB106. The rosette leaves of RNAi plants showed more severe permeable cuticle phenotypes than the myb106 mutants expressing the MYB16 amiRNA in the previous study. The RNAi plants also showed reduced expression of cuticle biosynthesis genes LACERATA and ECERIFERUM1. By contrast, expression of a gain-of-function MYB16 construct induced over-accumulation of waxy substances on leaves. These results suggest that MYB16 functions as a major regulator of cuticle formation in vegetative organs, in addition to its effect in reproductive organs and trichomes.     

Change of shoot architecture during juvenile-to-adult phase transition in soybean

Juvenile-to-adult phase change is an indispensable event which guarantees a successful life cycle. Phase change has been studied in maize, Arabidopsis and rice, but is mostly unknown in other species. Soybean/Fabaceae plants undergo drastic changes of shoot architecture at the early vegetative stage including phyllotactic change and leaf type alteration from simple to compound. These characteristics make soybean/Fabaceae plants an interesting taxon for investigating vegetative phase change. Following the expansion of two cotyledons, two simple leaves simultaneously emerge in opposite phyllotaxy. The phyllotaxy of the third and fourth leaves is not fixed; both opposite and distichous phyllotaxis are observed within the same population. Leaves were compound from the third leaf. But the third leaf was rarely simple. Morphological and quantitative changes in early vegetative phase were recognized in leaf size, leaf shape, number of trichomes, stipule size and shape, and shoot meristem shape. Two microRNA genes, miR156 and miR172, are known to be associated with vegetative phase change. Examination of the expression level revealed that miR156 expression was high in the first two leaves and subsequently down-regulated, and that of miR172 showed the inverse expression pattern. These expression patterns coincided with the case of other species. Taken all data together, the first and second leaves represent juvenile phase, the fifth and upper leaves adult phase, and the third and fourth leaves intermediate stage. Further investigation of soybean phase change would give fruitful understandings on plant development.     

Plant Responses to Brief Touching: A Mechanism for Early Neighbour Detection?

In natural habitats plants can be exposed to brief and light contact with neighbouring plants. This mechanical stimulus may represent a cue that induces responses to nearby plants. However, little is known about the effect of touching on plant growth and interaction with insect herbivores. To simulate contact between plants, a soft brush was used to apply light and brief mechanical stimuli to terminal leaves of potato Solanum tuberosum L. The number of non-glandular trichomes on the leaf surface was counted on images made by light microscope while glandular trichomes and pavement cells were counted on images made under scanning electronic microscope. Volatile compounds were identified and quantified using coupled gas chromatography–mass spectrometry (GC-MS). Treated plants changed their pattern of biomass distribution; they had lower stem mass fraction and higher branch and leaf mass fraction than untouched plants. Size, weight and number of tubers were not significantly affected. Touching did not cause trichome damage nor change their total number on touched terminal leaves. However, on primary leaves the number of glandular trichomes and pavement cells was significantly increased. Touching altered the volatile emission of treated plants; they released higher quantities of the sesquiterpenes (E)-β-caryophyllene, germacrene D-4-ol and (E)-nerolidol, and lower quantities of the terpenes (E)-ocimene and linalool, indicating a systemic effect of the treatment. The odour of touched plants was significantly less preferred by the aphids Macrosiphum euphorbiae and Myzus persicae compared to odour of untouched plants. The results suggest that light contact may have a potential role in the detection of neighbouring plants and may affect plant-insect interactions.     

Geographic association and temporal variation of chemical and physical defense and leaf damage in Datura stramonium

The evolution of plant defense traits has traditionally been explained trough the “coevolutionary arms race” between plants and herbivores. According to this, specialist herbivores have evolved to cope effectively with the defensive traits of their host plants and may even use them as a cue for host location. We analyzed the geographic association between leaf trichomes, two tropane alkaloids (putative resistance traits), and leaf damage by herbivores in 28 populations of Datura stramonium in central Mexico. Since the specialist leaf beetles Epitrix parvula and Lema trilineata are the main herbivores of D. stramonium in central Mexico, we predicted a positive association between plant defense and leaf damage across populations. Also, if physical environmental conditions (temperature or precipitation) constrain the expression of plant defense, then the geographic variation in leaf damage should be explained partially by the interaction between defensive traits and environmental factors. Furthermore, we studied the temporal and spatial variation in leaf trichome density and leaf damage in five selected populations of D. stramonium sampled in two periods (1997 vs. 2007). We found a positive association between leaf trichomes density and atropine concentration with leaf damage across populations. The interaction between defensive traits and water availability in each locality had a significant effect on the geographic variation in leaf damage. Differences among populations in leaf trichome density are maintained over time. Our results indicate that local plant–herbivore interaction plays an important role in shaping the geographic and temporal variation in plant defense in D. stramonium.     

Dimorphism in trichome production of <Emphasis Type="Italic">Persicaria lapathifolia</Emphasis> var. <Emphasis Type="Italic">lapathifolia</Emphasis> and Its multiple effects on a leaf beetle

Polymorphisms in plants are main factors that determine the diversity of associated animal communities and their population dynamics. Typically, Persicaria lapathifolia var. lapathifolia (Polygonaceae) has no trichomes on leaf surfaces (glabrous type), but a hairy type does sometimes occur. Based on a cultivation experiment, the presence or absence of trichomes is clarified to be under genetic control. To reveal the defensive function of trichomes against herbivores, laboratory experiments were conducted using a major herbivore, Galerucella grisescens (Coleoptera: Chrysomelidae). In both choice and no-choice feeding tests, the glabrous type was significantly more consumed by G. grisescens adults, while the hairy type was not consumed. In the hairy leaf treatment, larval duration tended to become longer, the adult body weight became significantly lower, and adults laid significantly more eggs than in the glabrous leaf treatment. Hairy leaves contained significantly more total phenolics and condensed tannins than glabrous leaves, suggesting that the hairy type allocates more resources for physical and chemical defence. Because no significant differences in leaf consumption were detected in the feeding experiment using powdered host leaves, G. grisescens seems to have adapted to the chemical defences of P. lapathifolia var. lapathifolia. These results clearly indicate that leaf trichomes of P. lapathifolia var. lapathifolia effectively act as a physical defence against G. grisescens.     

Arabidopsis ketoacyl‐CoA synthase 16 (KCS16) forms C36/C38 acyl precursors for leaf trichome and pavement surface wax

The aliphatic waxes sealing plant surfaces against environmental stress are generated by fatty acid elongase complexes, each containing a β‐ketoacyl‐CoA synthase (KCS) enzyme that catalyses a crucial condensation forming a new C─C bond to extend the carbon backbone. The relatively high abundance of C₃₅ and C₃₇ alkanes derived from C₃₆ and C₃₈ acyl‐CoAs in Arabidopsis leaf trichomes (relative to other epidermis cells) suggests differences in the elongation machineries of different epidermis cell types, possibly involving KCS16, a condensing enzyme expressed preferentially in trichomes. Here, KCS16 was found expressed primarily in Arabidopsis rosette leaves, flowers and siliques, and the corresponding protein was localized to the endoplasmic reticulum. The cuticular waxes on young leaves and isolated leaf trichomes of ksc16 loss‐of‐function mutants were depleted of C₃₅ and C₃₇ alkanes and alkenes, whereas expression of Arabidopsis KCS16 in yeast and ectopic overexpression in Arabidopsis resulted in accumulation of C₃₆ and C₃₈ fatty acid products. Taken together, our results show that KCS16 is the sole enzyme catalysing the elongation of C₃₄ to C₃₈ acyl‐CoAs in Arabidopsis leaf trichomes and that it contributes to the formation of extra‐long compounds in adjacent pavement cells.