烟草microRNA171c的功能分析

MicroRNA是一类长度为20~24碱基的非编码小RNA,调控植物多种生理代谢途径。MicroRNA171(miR171)在拟南芥、大麦和水稻中通过负调控SCL靶基因使植物表现出分枝结构变化和其他一些发育表型,还可调控拟南芥叶绿素的合成代谢。但其他植物miR171的功能仍然未知。为了探明烟草miR171c的功能,本研究根据烟草miR171c序列设计了靶基因模拟物STTM171,通过病毒表达载体在烟草中进行表达,抑制miR171c的活性后观察植物表型变化。结果表明在病毒表达STTM171烟草中,植株出现顶端优势丧失、茎干增多等表型。荧光定量PCR检测到STTM171过表达植株miR171c表达量下降,两个推测的SCL靶基因TC134811和TC127385表达量上升,表明miR171c可能在烟草和拟南芥等植物中的功能比较保守,可以通过调控可能的靶基因SCL来调节植物的生长发育。     

Arabidopsis protochlorophyllide oxidoreductase A (PORA) restores bulk chlorophyll synthesis and normal development to a porB porC double mutant

In angiosperms the strictly light-dependent reduction of protochlorophyllide to chlorophyllide is catalyzed by NADPH:protochlorophyllide oxidoreductase (POR). The Arabidopsis thaliana genome encodes three structurally related but differentially regulated POR genes, PORA, PORB and PORC. PORA is expressed primarily early in development—during etiolation, germination and greening. In contrast, PORB and PORC are not only expressed during seedling development but also throughout the later life of the plant, during which they are responsible for bulk chlorophyll synthesis. The Arabidopsis porB-1 porC-1 mutant displays a severe xantha (highly chlorophyll-deficient) phenotype characterized by smaller prolamellar bodies in etioplasts and decreased thylakoid stacking in chloroplasts. Here we have demonstrated the ability of an ectopic PORA overexpression construct to restore prolamellar body formation in the porB-1 porC-1 double mutant background. In response to illumination, light-dependent chlorophyll production, thylakoid stacking and photomorphogenesis are also restored in PORA-overexpressing porB-1 porC-1 seedlings and adult plants. An Arabidopsis porB-1 porC-1 double mutant can therefore be functionally rescued by the addition of ectopically expressed PORA, which suffices in the absence of either PORB or PORC to direct bulk chlorophyll synthesis and normal plant development.     

Chloroplast culture: The chlorophyll repair potential of mature chloroplasts incubated in a simple medium

The chlorophyll repair potential of mature Cucumis chloroplasts incubated in a simple Tris-HCI/sucrose medium is described. The chloroplasts were isolated from green, fully expanded Cucumis cotyledons which were capable of chlorophyll repair. This was evidenced by a functional chlorophyll biosynthetic pathway in the mature tissue. The biosynthesis of protochlorophyllide from exogenous δ-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between δ-aminolevulinic acid and protochlorophyllide. The conversion of exogenous protochlorophyllide into chlorophyll a was used as a marker for the operation of the chlorophyll pathway beyond protochlorophyllide. It appeared from these studies that contrary to published reports, unfortified fully developed Cucumis chloroplasts incubated in Tris-HCl/sucrose without the addition of cofactors exhibited a partial and limited chlorophyll repair capability. Their net tetrapyrrole biosynthetic competence from δ-aminolevulinic acid was confined to the accumulation of coproporphyrin. No net tetrapyrrole biosynthesis beyond coproporphyrin was observed. However, the plastids were capable of incorporating small amounts of δ-amino-[4-¹⁴C]levulinic acid into [¹⁴C] protochlorophyllide but were incapable of converting exogenous protochlorophyllide into chlorophyll. After prolonged incubation of the unfortified chloroplasts in the dark, a fluorescent protochlorophyllide-like compound accumulated. This compound [Cp (E₄₃₀-F₆₃₁)] exhibited a soret excitation maximum at 430 nm (E₄₃₀) and a fluorescence emission maximum at 631 nm (F₆₃₁) in methanol/acetone (4 : 1, v/v). Cp (E₄₃₀-F₆₃₁) was shown to be neither protochlorophyllide nor zinc-protochlorophyllide but an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.     

Analysis of biochemical variations and microRNA expression in wild (Ipomoea campanulata) and cultivated (Jacquemontia pentantha) species exposed to in vivo water stress

The current study analyses few important biochemical parameters and microRNA expression in two closely related species (wild but tolerant Ipomoea campanulata L. and cultivated but sensitive Jacquemontia pentantha Jacq.G.Don) exposed to water deficit conditions naturally occurring in the field. Under soil water deficit, both the species showed reduction in their leaf area and SLA as compared to well-watered condition. A greater decrease in chlorophyll was noticed in J. pentantha (~50 %) as compared to I. campanulata (20 %) under stress. By contrast, anthocyanin and MDA accumulation was greater in J. pentantha as compared to I. campanulata. Multiple isoforms of superoxide dismutases (SODs) with differing activities were observed under stress in these two plant species. CuZnSOD isoforms showed comparatively higher induction (~10–40 %) in I. campanulata than J. pentantha. MicroRNAs, miR398, miR319, miR395 miR172, and miR408 showed opposing expression under water deficit in these two plant species. Expression of miR156, miR168, miR171, miR172, miR393, miR319, miR396, miR397 and miR408 from either I. campanulata or J. pentantha or both demonstrated opposite pattern of expression to that of drought stressed Arabidopsis. The better tolerance of the wild species (I. campanulata) to water deficit could be attributed to lesser variations in chlorophyll and anthocyanin levels; and relatively higher levels of SODs than J. pentantha. miRNA expression was different in I. campanulata than J. pentantha.     

Light-dependent chlorophyll a biosynthesis upon chlL deletion in wild-type and photosystem I-less strains of the cyanobacterium Synechocystis sp. PCC 6803

Part of the chlL gene encoding a component involved in light-independent protochlorophyllide reduction was deleted in wild type and in a photosystem I-less strain of Synechocystis sp. PCC 6803. In resulting mutants, chlorophyll biosynthesis was fully light-dependent. When these mutants were propagated under light-activated heterotrophic growth conditions (in darkness except for 15 min of weak light a day) for several weeks, essentially no chlorophyll was detectable but protochlorophyllide accumulated. Upon return of the chlL ^- mutant cultures to continuous light, within the first 6 h chlorophyll was synthesized at the expense of protochlorophyllide at a rate independent of the presence of photosystem I. Chlorophyll biosynthesized during this time gave rise to a 685 nm fluorescence emission peak at 77 K in intact cells. This peak most likely originates from a component different from those known to be directly associated with photosystems II and I. Development of 695 and 725 nm peaks (indicative of intact photosystem II and photosystem I, respectively) required longer exposures to light. After 6 h of greening, the rate of chlorophyll synthesis slowed as protochlorophyllide was depleted. In the chlL ^- strain, greening occurred at the same rate at two different light intensities (5 and 50 E m^-2s^-1), indicating that also at low light intensity the amount of light is not rate-limiting for protochlorophyllide reduction. Thus, in this system the rate of chlorophyll biosynthesis is limited neither by biosynthesis of photosystems nor by the light-dependent protochlorophyllide reduction. We suggest the presence of a chlorophyll-binding chelator protein (with 77 K fluorescence emission at 685 nm) that binds newly synthesized chlorophyll and that provides chlorophyll for newly synthesized photosynthetic reaction centers and antennae.     

Chloroplast-encoded chlB gene from Pinus thunbergii promotes root and early chlorophyll pigment development in Nicotiana tabaccum

Chlorophyll biosynthesis is catalyzed by two multi subunit enzymes; a light-dependent and a light-independent protochlorophyllide oxidoreductase. The light-independent enzyme consists of three subunits (ChlL, ChlN and ChlB) in photosynthetic bacteria and plastids in which the chlB gene encodes the major subunit that catalyzes the reduction of protochlorophyllide to chlorophyllide. We report here stable integration of the chlB gene from Pinus thunbergii into the chloroplast genome of tobacco. Using helium-driven biolistic gun, transplastomic clones were developed in vitro. The stable integration and homoplasmy for transgenes was confirmed by using PCR and Southern blotting techniques. Nodal cuttings of the homoplasmic transgenic and untransformed wild type shoots were cultured on MS medium in the dark. As expected, shoots developed from the cuttings of the wild type plants in the dark showed etiolated growth with no roots whereas shoots from the cuttings of the transgenic plants developed early and more roots. Upon shifting from dark to light in growth room, leaves of the transgenic shoots showed early development of chlorophyll pigments compared to the wild type shoots. Further, photosynthetically indistinguishable transgenic shoots also showed significant difference in root development from untransformed wild type shoots when cuttings were grown in the light. Therefore, it may be concluded that the chlB gene is involved, directly or indirectly, in the root development of tobacco. Further, the gene promotes early development of chlorophyll pigments, upon illumination from dark, in addition to its role in the light-independent chlorophyll formation when expressed together with subunits L&N in other organisms.     

Biogenesis of photosynthetic apparatus under the inhibition of energy processes in de-etiolated seedlings of barley (Hordeum vulgare L.)

Transformation of protochlorophyllide forms in etiolated barley seedlings and biogenesis of photosynthetic apparatus in greening leaves of 7-day-old etiolated barley seedlings (Hordeum vulgare L.) were studied under the inhibition of energy processes during illumination. Repression of electron transport between photosystem 2 and 1 (PS2 and PS1, respectively) with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) inhibited the photochemical activity of PS2 but did not affect chlorophyll biosynthesis and ATP content in leaves compared to the control. Inhibition of mitochondrial electron transport with sodium azide increased relative content of nonphotoactive protochlorophyllide in etiolated leaves, decreased the content of ATP, chlorophylls, and carotenoids and completely suppressed the functional activity of PS 2. The inhibitor of glycolysis sodium fluoride affected all the parameters even more strongly. We observed synchronism in the accumulation of chlorophylls and carotenoids during greening for all inhibitor variants other than fluoride (correlation coefficient, r, equal to 0.98, 0.97, 0.97, and 0.47 with the significance level of 0.01; 0.015; 0.015, and 0.27 for control, diuron, azide, and sodium fluoride, respectively). The change in chlorophyll content under the influence of inhibitors positively correlated with the amount of ATP in the leaf tissue (for 24 h greening, r = 0.97 with significance level of 0.015). We suggest that sources of ATP involved in the synthesis of chlorophyll during greening of etiolated barley seedlings are mostly of non-plastid origin.