拟南芥矮小丛生突变体的分离与分子鉴定

顶端优势是指侧生分生组织的生长被主茎或主花序所抑制.最近的研究通过分离和鉴定顶端优势发生改变的突变体开始揭示顶端优势的分子机制.通过T-DNA标签法分离了拟南芥矮小丛生(bushy and dwarf 1, bud1 )突变体.突变体植株的表型包括顶端优势丧失、株型矮小,表明bud1 突变体存在生长素代谢、运输或信号传导的缺陷.一个对生长素特异反应的启动子驱动的报告基因在bud1 中表达模式改变.生长素敏感性和运输能力的测定表明这两个过程在 bud1中均正常.以上结果显示bud1 表型是生长素代谢缺陷的结果.遗传分析表明BUD1 为半显性突变且与一个T-DNA插入共分离,可通过iPCR方法分离.     

拟南芥矮小丛生突变体的分离与分子鉴定

顶端优势是指侧生分生组织的生长被主茎或主花序所抑制。最近的研究通过分离和鉴定顶端优势发生改变的突变体开始揭示顶端优势的分子机制。通过T-DNA标签法分离了拟南芥矮小丛生(bushy and dwarf l,budl)突变体。突变体植株的表型包括顶端优势丧失、株型矮小,表明budl突变体存在生长素代谢、运输或信号传导的缺陷。一个对生长素特异反应的启动子驱动的报告基因在budl中表达模式改变。生长素敏感性和运输能力的测定表明这两个过程在budl中均正常。以上结果显示budl表型是生长素代谢缺陷的结果。遗传分析表明BUDI为半显性突变且与一个T-DNA插入共分离,可通过iPCR方法分离。     

Increased expression of MAP KINASE KINASE7 causes deficiency in polar auxin transport and leads to plant architectural abnormality in Arabidopsis

Polar auxin transport (PAT) plays a crucial role in the regulation of many aspects of plant growth and development. We report the characterization of a semidominant Arabidopsis thaliana bushy and dwarf1 (bud1) mutant. Molecular genetic analysis indicated that the bud1 phenotype is a result of increased expression of Arabidopsis MAP KINASE KINASE7 (MKK7), a member of plant mitogen-activated protein kinase kinase group D. We showed that BUD1/MKK7 is a functional kinase and that the kinase activity is essential for its biological functions. Compared with the wild type, the bud1 plants develop significantly fewer lateral roots, simpler venation patterns, and a quicker and greater curvature in the gravitropism assay. In addition, the bud1 plants have shorter hypocotyls at high temperature (29 degrees C) under light, which is a characteristic feature of defective auxin action. Determination of tritium-labeled indole-3-acetic acid transport showed that the increased expression of MKK7 in bud1 or the repressed expression in MKK7 antisense transgenic plants causes deficiency or enhancement in auxin transport, indicating that MKK7 negatively regulates PAT. This conclusion was further substantiated by genetic and phenotypic analyses of double mutants generated from crosses between bud1 and the auxin-related mutants axr3-3, tir1-1, doc1-1, and atmdr1-1.     

Roles for Auxin, Cytokinin, and Strigolactone in Regulating Shoot Branching

Many processes have been described in the control of shoot branching. Apical dominance is defined as the control exerted by the shoot tip on the outgrowth of axillary buds, whereas correlative inhibition includes the suppression of growth by other growing buds or shoots. The level, signaling, and/or flow of the plant hormone auxin in stems and buds is thought to be involved in these processes. In addition, RAMOSUS (RMS) branching genes in pea (Pisum sativum) control the synthesis and perception of a long-distance inhibitory branching signal produced in the stem and roots, a strigolactone or product. Auxin treatment affects the expression of RMS genes, but it is unclear whether the RMS network can regulate branching independently of auxin. Here, we explore whether apical dominance and correlative inhibition show independent or additive effects in rms mutant plants. Bud outgrowth and branch lengths are enhanced in decapitated and stem-girdled rms mutants compared with intact control plants. This may relate to an RMS-independent induction of axillary bud outgrowth by these treatments. Correlative inhibition was also apparent in rms mutant plants, again indicating an RMS-independent component. Treatments giving reductions in RMS1 and RMS5 gene expression, auxin transport, and auxin level in the main stem were not always sufficient to promote bud outgrowth. We suggest that this may relate to a failure to induce the expression of cytokinin biosynthesis genes, which always correlated with bud outgrowth in our treatments. We present a new model that accounts for apical dominance, correlative inhibition, RMS gene action, and auxin and cytokinin and their interactions in controlling the progression of buds through different control points from dormancy to sustained growth.     

Overexpression of a NAC-domain protein promotes shoot branching in rice

For a better understanding of shoot branching in rice (Oryza sativa), a rice activation-tagging library was screened for mutations in tiller development. Here, an activation-tagging mutant Ostil1 (Oryza sativa tillering1) was characterized, which showed increased tillers, enlarged tiller angle and semidwarf phenotype. Flanking sequence was obtained by plasmid rescue. RNA-interfering and overexpression transgenic rice plants were produced using Agrobacterium-mediated transformation. The mutant phenotype was cosegregated with the reallocation of Ds element, and the flanking region of the reallocated Ds element was identified as part of the OsNAC2 gene. Northern analysis showed that expression of OsNAC2 was greatly induced in the mutant plants. Transgenic rice overexpressing the OsNAC2 resulted in recapture of the mutant phenotype, while downregulation of OsNAC2 in the Ostil1 mutant through RNA interfering (RNAi) complemented the mutant phenotype, confirming that the Ostil1 was caused by overexpression of OsNAC2. Overexpression of OsNAC2 regulates shoot branching in rice. Overexpression of OsNAC2 contributes tiller bud outgrowth, but does not affect tiller bud initiation. This suggests that OsNAC2 has potential utility for improving plant structure for higher light-use efficiency and higher yield potential in rice.     

A COMPARATIVE DEVELOPMENTAL STUDY OF THE MUTANT SIDESHOOTLESS AND NORMAL TOMATO PLANTS

'Sideshootless,’ a mutant strain of tomato which does not produce axillary buds during vegetative growth, was compared with normally branching plants in order to study the nature of development particularly with regard to axillary buds. Sectioned material revealed no indication of axillary bud initiation in the sideshootless plant at any time during the vegetative phase of growth. In the normal plants, buds were noted to arise in the axil of the fifth youngest leaf. The buds take their origin in tissue which is in direct continuity with the apical meristem. The bud primordia later become set apart from the apex as vacuolation takes place in the surrounding tissue. At the time of floral initiation, the mutant and normal strains behave similarly. Axillary buds appear in the axils of the 2 leaves immediately below the floral apex. One of the buds elongates to overtop the existing plant axis; the other develops as a typical sidebranch. The inflorescence is pushed aside in the process. This pattern is repeated with each inflorescence; thus an axis composed of several superimposed laterals results.     

Proteomic Analysis Points to a Role for RAD23 in Apical Dominance in Pinus sylvestris var. Mongolica

In higher plants, apical dominance is the phenomenon whereby the main central stem is dominant over other side stems. In the present study, the natural loss of apical dominance in a Pinus sylvestris var. mongolica mutant was characterized. The mutant lacked an obvious trunk, but exhibited a large number of side stems, short internodes and leaves, and dwarfism. Cell biological analyses indicated that the meristem was changed dramatically in the mutant, with irregular cell arrangements, and no obvious layers of meristem and mature tissue. The loss of apical dominance in the mutant plant was accompanied by a significant decrease in auxin (A) and cytokinin (C) content compared to wild type, and increased C/A values. Proteomic analysis of the apical buds using 2DE-ESI-MS/MS at different stages of development (including dormancy, bud break, 10 days after bud break, and 15 days after bud break) revealed 49 proteins that were significantly changed in abundance compared to wild type. These proteins were involved in stress response, energy production and conversion, post-translation modification and amino acid transport and metabolism, as well as many other complex biological processes. Among the identified proteins, Rad23, an abscisic acid- and stress-induced protein, and cytoplasmic aconitate hydratase likely participate in ABA responses, Fe²⁺ ion homeostasis and the ubiquitin/26S proteasome pathway to regulate apical dominance of buds. Rad23, in particular, appears likely to play an important role in the complicated mechanism of apical dominance of Pinus sylvestris var. mongolica. This work provides important insights into this process as well as forming a basis for the further study of plant apical dominance.     

amp1 - a mutant with high cytokinin levels and altered embryonic pattern, faster vegetative growth, constitutive photomorphogenesis and precocious flowering

amp1 , a mutant of Arabidopsis thaliana has a phenotype altered in three different aspects of plant development; spatial pattern, photomorphogenetic growth, and initiation of flowering. While fewer than 0.1% of the seedlings of wild-type plants are non-dicot as many as 20% of the seedlings of the amp1 mutant are tricot or tetracot. The rate of leaf initiation is faster and vegetative phyllotaxy is altered in amp1 . When grown in the dark amp1 seedlings show morphogenetic properties similar to light-grown wild-type plants: they do not form an apical hook, have hypocotyls shorter than wild-type plants and form etiolated true leaves. amp1 mutant flowers significantly earlier than congenic Amp1 plants. The mutant has six times more cytokinin than wild-type suggesting that endogenous cytokinin levels might play an important role in mediating these different developmental processes. AMP1 might code for a negative regulator of cytokinin biosynthesis, or may be required for the degradation of cytokinin.     

Developmental defects in mutants of the PsbP domain protein 5 in Arabidopsis thaliana

Plants contain an extensive family of PsbP-related proteins termed PsbP-like (PPL) and PsbP domain (PPD) proteins, which are localized to the thylakoid lumen. The founding member of this family, PsbP, is an established component of the Photosystem II (PS II) enzyme, and the PPL proteins have also been functionally linked to other photosynthetic processes. However, the functions of the remaining seven PPD proteins are unknown. To elucidate the function of the PPD5 protein (At5g11450) in Arabidopsis, we have characterized a mutant T-DNA insertion line (SALK_061118) as well as several RNAi lines designed to suppress the expression of this gene. The functions of the photosynthetic electron transfer reactions are largely unaltered in the ppd5 mutants, except for a modest though significant decrease in NADPH dehydrogenase (NDH) activity. Interestingly, these mutants show striking plant developmental and morphological defects. Relative to the wild-type Col-0 plants, the ppd5 mutants exhibit both increased lateral root branching and defects associated with axillary bud formation. These defects include the formation of additional rosettes originating from axils at the base of the plant as well as aerial rosettes formed at the axils of the first few nodes of the shoot. The root-branching phenotype is chemically complemented by treatment with the synthetic strigolactone, GR24. We propose that the developmental defects observed in the ppd5 mutants are related to a deficiency in strigolactone biosynthesis.     

A Novel Family of Magnesium Transport Genes in Arabidopsis

Magnesium (Mg(2+)) is the most abundant divalent cation in plant cells and plays a critical role in many physiological processes. We describe the identification of a 10-member Arabidopsis gene family (AtMGT) encoding putative Mg(2+) transport proteins. Most members of the AtMGT family are expressed in a range of Arabidopsis tissues. One member of this family, AtMGT1, functionally complemented a bacterial mutant lacking Mg(2+) transport capability. A second member, AtMGT10, complemented a yeast mutant defective in Mg(2+) uptake and increased the cellular Mg(2+) content of starved cells threefold during a 60-min uptake period. (63)Ni tracer studies in bacteria showed that AtMGT1 has highest affinity for Mg(2+) but may also be capable of transporting several other divalent cations, including Ni(2+), Co(2+), Fe(2+), Mn(2+), and Cu(2+). However, the concentrations required for transport of these other cations are beyond normal physiological ranges. Both AtMGT1 and AtMGT10 are highly sensitive to Al(3+) inhibition, providing potential molecular targets for Al(3+) toxicity in plants. Using green fluorescence protein as a reporter, we localized AtMGT1 protein to the plasma membrane in Arabidopsis plants. We suggest that the AtMGT gene family encodes a Mg(2+) transport system in higher plants.     

Dose-Dependent Effect of Salicylates in a Moss, Funaria hygrometrica

Plant growth regulators now include more than the classic examples auxin, cytokinin, ethylene, and gibberellin, but little is known about the activity of these additional classes of molecules in nonvascular plants. The formation of buds by protonema of the moss Funaria hygrometrica is perhaps the best known and most fully characterized developmental system in the nonvascular plants. Examination of the effects of exogenously supplied salicylic acid and acetylsalicylate on this bioassay system shows that salicylates can regulate growth and development in mosses, producing a dose-dependent inhibition of bud formation. Other experiments show that this action is distinct from any direct effect on the well-known cytokinin stimulation of bud formation, occurs late in the process of bud formation, occurs prior to the stable commitment of nascent buds, and is not an effect on the outgrowth of young shoots. Because mosses are the sister clade to the vascular plants, these results suggest that the ability to perceive and transduce salicylate signals is an ancient feature of land plant physiology.     

A developmentally linked, dramatic, and transient loss of virus from roots of Arabidopsis thaliana plants infected by either of two RNA viruses

Possible effects of host developmental stage on the amount of virus present in systemically infected plant tissues hitherto have received little attention. In this study, the pattern of virus accumulation over the plant lifespan has been examined in systemically invaded tissues of Arabidopsis thaliana infected by either of two distinct (+)RNA viruses: Turnip mosaic virus, a member of Potyvirus, and Oilseed rape mosaic virus, a member of Tobamovirus. Quantitative analyses of virus coat protein and virus genomic RNA in roots versus aerial plant parts revealed generally sinusoidal temporal patterns of virus accumulation. In noninoculated leaves, a time period was found during which no virus accumulation was detected. This period was coincident with the approximately 7 days of inflorescence bud formation and differentiation. In roots, virion content reached high levels a few days after inoculation, dropping dramatically during the period of bud formation and quickly recovering after it. These results, together with electron microscopy observations, are consistent with loss of virions due to disassembly. Fluorescence observations of green fluorescent protein-tagged virus-infected root tissue also were consistent with a net loss of virus-specified proteins. Inoculations performed after the emergence of the inflorescence and on A. thaliana flowering-time mutants support the temporal link between observed changes in virus content and inflorescence bud formation. Different host-involving biochemical processes can be invoked to provide mechanistic clues, but no one of them alone seems sufficient to explain the complex patterns of tight temporal regulation of virus accumulation observed in these experiments.     

A plasma membrane-bound putative endo-1,4-beta-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis.

Endo-1,4-beta-D-glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non-crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane-anchored member of the EGase family, which is highly conserved between mono- and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark-grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR-mRNA levels. However, reduced KOR-mRNA levels were observed in det2, a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose-hemicellulose network in the expanding cell wall.     

DWARF27, an Iron-Containing Protein Required for the Biosynthesis of Strigolactones,Regulates Rice Tiller Bud Outgrowth

Tillering in rice (Oryza sativa) is one of the most important agronomic traits that determine grain yields. Previous studies on rice tillering mutants have shown that the outgrowth of tiller buds in rice is regulated by a carotenoid-derived MAX/RMS/D (more axillary branching) pathway, which may be conserved in higher plants. Strigolactones, a group of terpenoid lactones, have been recently identified as products of the MAX/RMS/D pathway that inhibits axillary bud outgrowth. We report here the molecular genetic characterization of d27, a classic rice mutant exhibiting increased tillers and reduced plant height. D27 encodes a novel iron-containing protein that localizes in chloroplasts and is expressed mainly in vascular cells of shoots and roots. The phenotype of d27 is correlated with enhanced polar auxin transport. The phenotypes of the d27 d10 double mutant are similar to those of d10, a mutant defective in the ortholog of MAX4/RMS1 in rice. In addition, 2′-epi-5-deoxystrigol, an identified strigolactone in root exudates of rice seedlings, was undetectable in d27, and the phenotypes of d27 could be rescued by supplementation with GR24, a synthetic strigolactone analog. Our results demonstrate that D27 is involved in the MAX/RMS/D pathway, in which D27 acts as a new member participating in the biosynthesis of strigolactones.     

Mutations in LACS2, a long-chain acyl-coenzyme A synthetase, enhance susceptibility to avirulent Pseudomonas syringae but confer resistance to Botrytis cinerea in Arabidopsis

We identified an Arabidopsis (Arabidopsis thaliana) mutant, sma4 (symptoms to multiple avr genotypes4), that displays severe disease symptoms when inoculated with avirulent strains of Pseudomonas syringae pv tomato, although bacterial growth is only moderately enhanced compared to wild-type plants. The sma4 mutant showed a normal susceptible phenotype to the biotrophic fungal pathogen Erysiphe cichoracearum. Significantly, the sma4 mutant was highly resistant to a necrotrophic fungal pathogen, Botrytis cinerea. Germination of B. cinerea spores on sma4 mutant leaves was inhibited, and penetration by those that did germinate was rare. The sma4 mutant also showed several pleiotropic phenotypes, including increased sensitivity to lower humidity and salt stress. Isolation of SMA4 by positional cloning revealed that it encodes LACS2, a member of the long-chain acyl-CoA synthetases. LACS2 has previously been shown to be involved in cutin biosynthesis. We therefore tested three additional cutin-defective mutants for resistance to B. cinerea: att1 (for aberrant induction of type three genes), bodyguard, and lacerata. All three displayed an enhanced resistance to B. cinerea. Our results indicate that plant cutin or cuticle structure may play a crucial role in tolerance to biotic and abiotic stress and in the pathogenesis of B. cinerea.     

Strigolactone may interact with gibberellin to control apical dominance in pea (Pisum sativum)

The role of strigolactones as plant growth regulators has been demonstrated through research on biosynthesis and signaling mutant plants and through the use of GR24, a synthetic analog of this class of molecules. Strigolactone mutants show a bushy phenotype and GR24 application inhibits the growth of axillary buds in these mutants, thus restoring the phenotype of a wild plant, which is characterized by a stronger apical dominance. In this work, we tested the effectiveness of this chemical on pea (Pisum sativum) plants following apex removal, which disrupts apical dominance and leads to axillary bud outgrowth. Moreover, we searched for relationships between the response to the strigolactone and gibberellin metabolism by applying GR24 to both climbing and dwarf peas, the latters being mutants for gibberellin biosynthesis. The results suggest that the endogenous level of the bioactive gibberellin GA₁ might modulate the response of decapitated pea plants to GR24, by changing bud sensitivity to the applied strigolactone.