Biological and biochemical characterization of a red-eye mutant in Nilaparvata lugens （Hemiptera： Delphacidae）

A red-eye colony was established in our laboratory in brown planthopper （BPH）, Nilaparvata lugens （Stal）, a major rice pest in Asia. Except for the red-eye phenotype, no other differences were observed between the wild-type （brown eye） and the mutant-type （red eye） in external characters. Genetic analysis revealed that the red-eye phenotype was controlled by a single autosomal recessive allele. Biological studies found that egg produc- tion and egg viability in the red-eye mutant colony were not significantly different from those in the wild-type BPH. Biochemical analysis and electronic microscopy examination revealed that the red-eye mutants contained decreased levels of both xanthommatin （brown） and pteridine （red） and reduced number of pigment granules. Thus, the changes of amount and ratio of the two pigments is the biochemical basis of this red-eye mutation. Our results indicate that the red-eye mutant gene （red） might be involved in one common gene locus shared by the two pigments in pigment transportation, pigment granule formation or some other processes.     

A DTX/MATE-Type Transporter Facilitates Abscisic Acid Efflux and Modulates ABA Sensitivity and Drought Tolerance in Arabidopsis

Abscisic acid （ABA） regulates numerous physiological and developmental processes in plants. Recent studies identify intracellular ABA receptors, implicating the transport of ABA across cell membranes as crucial for ABA sensing and response. Here, we report that a DTX/Multidrug and Toxic Compound Extrusion （MATE） family member in Arabidopsis thaliana, AtDTX50, functions as an ABA efflux transporter. When expressed heterologously in both an Escherichia coli strain and Xenopus oocyte cells, AtDTX50 was found to facilitate ABA efflux. Furthermore, dtx50 mutant mesophyll cells preloaded with ABA released less ABA compared with the wild-type （WT）. The AtDTX50 gene was expressed mainly in the vascular tissues and guard ceils and its expression was strongly up-regulated by exogenous ABA. The AtDTX50：：GFP fusion protein was localized predominantly to the plasma membrane. The dtx50 mutant plants were observed to be more sensitive to ABA in growth inhibition. In addition, compared with the WT, dtx50 mutant plants were more tolerant to drought with lower stomatal conductance, consistent with its function as an ABA efflux carrier in guard cells.     

Perturbation of Auxin Homeostasis Caused by Mitochondrial FtSH4 Gene-Mediated Peroxidase Accumulation Regulates Arabiclopsis Architecture

Reactive oxygen species and auxin play important roles in the networks that regulate plant development and morphogenetic changes, However, the molecular mechanisms underlying the interactions between them are poorly understood. This study isolated a mas （More Axillary Shoots） mutant, which was identified as an allele of the mitochondrial AAA-protease AtFtSH4, and characterized the function of the FtSH4 gene in regulating plant development by medi- ating the peroxidase-dependent interplay between hydrogen peroxide （H2Oz） and auxin homeostasis. The phenotypes of dwarfism and increased axillary branches observed in the mas （renamed as ftsh4-4） mutant result from a decrease in the IAA concentration. The expression levels of several auxin signaling genes, including IAA1, IAA2, and IAA3, as well as several auxin binding and transport genes, decreased significantly in ftsh4-4 plants. However, the H202 and peroxidases levels, which also have IAA oxidase activity, were significantly elevated in ftsh4-4 plants. The ftsh4-4 phenotypes could be reversed by expressing the iaaM gene or by knocking down the peroxidase genes PRX34 and PRX33. Both approaches can increase auxin levels in the ftsh4-4 mutant. Taken together, these results provided direct molecular and genetic evidence for the interaction between mitochondrial ATP-dependent protease, H2O2, and auxin homeostasis to regulate plant growth and development.     

A Novel ABA Insensitive Mutant of Lotus japonicus with a Wilty Phenotype Displays Unaltered Nodulation Regulation

An ABA insensitive mutant, Beyma, was isolated in Lotus japonicus MG-20 from an EMS mutagenesis population using root growth inhibition to applied ABA as the screening criterion. （The name ＂Beyma＂ was taken from the Australian Aboriginal language, Wagiman, beyma, meaning ‘drying up＇.） The stable mutant that segregates as a dominant Mendelian mutation is insensitive to ABA induced inhibition of germination, vegetative growth, stomatal opening, as well as nodulation. Tissue ABA levels were normal, suggesting a sensitivity rather than biosynthesis mutation. It is slow-growing （50-70% of wild-type MG-20） and has a near-constitutive wilty phenotype associated with its inability to regulate stomatal opening. Whilst showing a wide range of ABA insensitive phenotypes, Beyma did not show alteration of nodule number control, as, in the absence of added ABA, the number and patterning （but not size） of nodules formed in the mutant were similar to that of MG-20. Split root experiments on MG-20 showed that application of ABA on one side of the root inhibited nodulation locally but not systemically. We propose that ABA is not involved directly in systemic autoregulation of nodulation （AON）.     

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice （Oryza sativa L.）

Gibberellin (GA) 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs (GA3 or GA4) can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs (GA12 and/or GA53) as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.     

Evidences of Hfq associates with tryptophanase and affects extracellular indole levels

In this study, we observed a novel property of Escherichia coli Hfq protein： it possibly influenced extracellular indole levels. The extracellular indole concentrations were increased in Hfq mutant cells and decreased in Hfq overexpression cells in a cell densitydependent manner. The decreased extracellular indole levels in Hfq overexpression cells caused the postponement of entering into stationary phase. Indole was produced by tryptophanase, the gene product of tnaA, which catalyzed tryptophan into indole, ammonia and pyruvate. Further studies showed that at cell density of 0.8 but not at 0.4, tryptophanase activities of total cell extracts were affected by Hfq mutation or overexpression. Protein puff-down assay and co-immunoprecipitation experiments revealed that Hfq associated with tryptophanase under relatively higher extracellular indole levels, suggesting this was a feedback control of indole production. The association of Hfq and tryptophanase might be indirect because purified Hfq could not affect the values of Km and Vmax of purified tryptophanase.     

Production of Polyamines Is Enhanced by Endogenous Abscisic Acid in Maize Seedlings Subjected to Salt Stress

It is known that salt stress and exogenously applied abscisic acid （ABA） can enhance the polyamine content in plants and that salt stress itself can lead to an increase in endogenous ABA production. In the present study, the relationships between salt-induced ABA and polyamine accumulation were inves- tigated using ABA-deficient mutant （vp5/vp5） maize （Zea mays L.） seedlings and ABA and polyamine biosynthesis inhibitors. The results show that reduced endogenous ABA levels, as a result of either the mutation or by using a chemical inhibitor （sodium tungstate）, also reduced the accumulation of polyamines in salt-stressed leaves of maize seedlings. The polyamine synthesis inhibitors D-arginine and α- difluoromethylornithine also reduced the polyamine content of the leaves of maize seedling under salt stress. Both ABA and polyamine enhanced the dry weight accumulation of salt-stressed seedlings and also increased the activities of the two dominant tonoplast membrane enzymes, H^＋-ATPase and H^＋-PPase, when plants were under salt stress. The results suggest that salt stress induces an increase in endogenous ABA levels, which then enhances polyamine synthesis. Such responses may increase a plant＇s tolerance to salt.     

The Arabidopsis Spontaneous Cell Death1 gene, encoding a ζ-carotene desaturase essential for carotenoid biosynthesis, is involved in chloroplast development,photoprotection and retrograde signalling

Carotenoids, a class of natural pigments found in all photosynthetic organisms, are involved in a variety of physiological processes, including coloration, photoprotection, biosynthesis of abscisic acid （ABA） and chloroplast biogenesis. Although carotenoid biosynthesis has been well studied biochemically, the genetic basis of the pathway is not well understood. Here, we report the characterization of two allelic Arabidopsis mutants, spontaneous cell death1-1 （spcl-1） and spc1-2. The weak allele spc1-1 mutant showed characteristics of bleached leaves, accumulation of superoxide and mosaic cell death. The strong mutant allele spc1-2 caused a complete arrest of plant growth and development shortly after germination, leading to a seedling-lethal phenotype. Genetic and molecular analyses indicated that SPC1 encodes a putative ζ-carotene desaturase （ZDS） in the carotenoid biosynthesis pathway. Analysis of carotenoids revealed that several major carotenoid compounds downstream of SPC 1/ZDS were substantially reduced in spc1-1, suggesting that SPC 1 is a functional ZDS. Consistent with the downregulated expression of CAO and PORB, the chlorophyll content was decreased in spc1-1 plants. In addition, expression of Lhcb1. 1, Lhcbl. 4 and RbcS was absent in spc1-2, suggesting the possible involvement of carotenoids in the plastid-to-nucleus retrograde signaling. The spc1-1 mutant also displays an ABA-deficient phenotype that can be partially rescued by the externally supplied phytohormone. These results suggest that SPC1/ZDS is essential for biosynthesis of carotenoids and plays a crucial role in plant growth and development.     

Cell wall invertase-deficient miniature1 kernels have altered phytohormone levels

The Zea mays (maize) miniature1 (Mn1) locus encodes the cell wall invertase INCW2, which is localized predominantly in the basal endosperm transfer layer (BETL) of developing kernels and catalyzes conversion of sucrose into glucose and fructose. Mutations in Mn1 result in numerous changes that include a small kernel phenotype resulting from both decreased cell size and number. To explore the pleiotropic effects of this mutation, we investigated the levels of indole-3-acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) in basal regions, upper regions, and embryos of developing kernels in the inbred line W22. We measured phytohormones from 6 to 28 days after pollination (DAP) in wild type (WT) and two alleles of mn1, mn1-1 and mn1-89. IAA was the predominant hormone in kernels, with WT levels of free IAA accumulating over time to more than 2microg/g of fresh weight. Kernels of mn1-1 accumulated up to 10-fold less IAA than WT, and levels of IAA sugar conjugates were similarly reduced. Although less abundant, differences were also observed in levels of ABA, JA, and SA between WT and the mn1 alleles. SA levels were increased by as much as 10-fold in mn1-1, and mn1-89 displayed intermediate SA levels at most timepoints. These findings indicate that invertase-mediated sucrose cleavage directly or indirectly regulates the levels of key plant hormones during seed development.     

The bushy pea mutant is IAA-deficient

The bushy mutant of pea ( Pisum sativum L.) is characterized by short, thin stems, very small leaves and profuse branching. The bushy phenotype is conferred by a dominant allele, termed bsh . Here we show that bushy plants contain lower levels of free indole-3-acetic acid (IAA) than wild-type (WT) plants. On emergence from the growth medium, bushy seedlings do not immediately display the mutant phenotype, and the effect of bsh on IAA content is small. After 10–14 days, the bushy phenotype begins to develop, and while the IAA content rises in WT plants, it falls in the mutant. The resulting difference in IAA level between WT and mutant can be up to 12-fold. Although there is a deficiency of free IAA in bushy plants, the total IAA content (including free and conjugated forms) was not reduced in comparison with the WT. Furthermore, in bushy plants, the level of the main IAA conjugate in pea, IAAsp, was not reduced to the same extent as that of IAA, and metabolism studies indicated that faster IAA deactivation might contribute to IAA deficiency in the mutant. Application of IAA to bushy plants did not result in a WT phenocopy. However, the short internodes and profuse branching of bushy plants is consistent with classical views on how IAA affects plant development.     

Light quality regulation of endogenous levels of auxin,abscisic acid and ethylene production in petioles and leaves of wild type and ACC deaminase transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> seedlings

Brassica napus L. seedlings responded to low red to far-red (R/FR) ratio by elongating petioles and decreasing leaf expansion. These typical shade avoidance traits were correlated with significantly decreased endogenous indole-3-acetic acid (IAA) levels and significantly increased endogenous abscisic acid (ABA) levels and ethylene production. The transgenic (T) B. napus line bearing the bacterial ACC deaminase gene, did not respond to low R/FR ratio with altered petiole and leaf growth and less ethylene (especially by petioles) was produced. As with WT seedlings, T seedlings had significantly lower IAA levels in both petioles and leaves under low R/FR ratio. However, ABA levels of low R/FR ratio-grown T seedlings either increased (petioles) or were unaltered (leaves). Our results further suggest that low R/FR ratio regulates endogenous IAA levels independently of ethylene, but there may be an interaction between ABA and ethylene in leaf development.     

Reduced de-etiolation of hypocotyl growth in a tomato mutant is associated with hypersensitivity to, and high endogenous levels of, abscisic acid.

A recessive single gene mutant, 7B-1, in tomato was originally selected for its photoperiod-dependent male sterility. The 7B-1 mutant also has some pleiotropic effects including reduced light-induced inhibition, i.e. de-etiolation, of the hypocotyl in long days (LD), increased seed size and weight, and reduced transpiration rate. These traits led us to investigate the sensitivity of 7B-1 to exogenous hormones and the interaction of these responses with daylength. In LD, but not in short days (SD), 7B-1 was more sensitive than wild-type (WT) to exogenous abscisic acid (ABA) for inhibition of seed germination, root elongation and transpiration rate. 7B-1 mutant also exhibited reduced responses to exogenous gibberellin (GA(3)) for hypocotyl elongation, and to inhibitors of GA biosynthesis for seed germination and root and hypocotyl elongation. 7B-1 hypocotyls contained a higher level of endogenous ABA than WT in both photoperiods, although ABA levels were higher in LD than in SD. In contrast, growth-active GAs, i.e. GA(1), GA(3) and GA(4), and IAA were low in the mutant hypocotyls. The 7B-1 mutant appears to be an ABA-overproducer, and the photoperiod-regulated ABA levels may be responsible for the hypersensitivity of the mutant to exogenous ABA.     

Fluctuation in levels of endogenous plant hormones in ovules of normal and mutant cotton during flowering and their relation to fiber development

The contents of indole-3-acetic acid (IAA), gibberellins (GAs), abscisic acid (ABA), and cytokinins were determined in ovules of normal cotton (Tm-1) and a kind of fiber differentiation mutant (Xin) before and after flowering by enzyme-linked immunosorbent assays. It was found that 24 h before flowering, a peak of IAA content was observed in ovules of Tm-1, whereas in ovules of Xin, a low level of IAA was determined. From –1 day (1 day before flowering) to +3 days (3 days after flowering), GA₁₊₃ levels in ovules of Xin were 40–70% lower than those of Tm-1; GA₄₊₇ levels were very low, and there was no visible difference in GA₄₊₇ content between normal and mutant cotton. The ABA content in ovule of Tm-1 decreased by 70% 3 days after flowering, whereas that of Xin only decreased by 20%. The levels of cytokinins in ovules of Tm-1 decreased after flowering, and those of Xin kept up a steady increase.Abbreviations IAA indole-3-acetic acid - GA gibberellin - ABA abscisic acid - ELISA enzyme-linked immunosorbent assay - FW fresh weight - PBS phosphate-buffered saline - iPA isopentenyladenosine - ZR zeatin riboside - DHZR dihydrozeatin riboside - CTK cytokinin     

Stems of the <Emphasis Type="Italic">Arabidopsis pin1-1</Emphasis> mutant are not deficient in free indole-3-acetic acid

The pin1-1 mutant of Arabidopsis thaliana has been pivotal for studies on auxin transport and on the role of auxin in plant development. It was reported previously that when whole shoots were analysed, levels of the major auxin, indole-3-acetic acid (IAA) were dramatically reduced in the mutant, compared with the WT (Okada et al. 1991). The cloning of PIN1, however, provided evidence that this gene encodes a facilitator of auxin efflux, raising the question of how the pin1-1 mutation might reduce overall IAA levels as well as IAA transport. We therefore re-examined IAA levels in individual parts of pin1-1 and WT plants, focusing on inflorescence stems. Our data show that there is in fact no systemic IAA deficiency in the mutant. The previously reported difference between mutant and WT may have been due to the inclusion of reproductive structures in the WT harvest: we show here that the inflorescence itself contains high levels of IAA. We reconcile the normal IAA levels of pin1-1 inflorescence stems with their (previously-reported) reduced ability to transport IAA by presenting evidence that the auxin in mutant stems is not imported from their apical portion. Our data also indicate that levels of another auxin, indole-3-butyric acid (IBA), are very low in stems of the genotypes used in this study.     

NaCI Reduces Indole-3-Acetic Acid Levels in the Roots of Tomato Plants Independent of Stress-Induced Abscisic Acid

Indole-3-acetic acid (IAA) was measured in leaves and roots of tomato (Lycopersicon esculentum) genotypes subjected to salt stress. An abscisic acid (ABA)-deficient mutant of tomato (sitiens), the genetic parent (Rheinlands Ruhm, RR), and a commercial variety (Large Cherry Red, LCR) of tomato were treated with 50 to 300 mM NaCl in nutrient culture. Both LCR and RR had significantly higher levels of IAA in the roots compared with that in sitiens prior to treatment. The initial levels of IAA in the roots of LCR and RR declined by nearly 75% after exposure to NaCl, whereas those in roots from the sitiens mutant remained unchanged. IAA levels in the leaves of all genotypes remained unchanged or increased slightly in response to NaCl. ABA was highest in leaves from the normal genotypes after exposure to NaCl. ABA levels in the roots of sitiens were similar to the levels in the normal genotypes, whereas levels in the leaves were only 10% of the levels found in normal genotypes regardless of the salt treatment. Treatment of LCR and sitiens with exogenous ABA increased the ABA levels in leaves and roots, but there were no measurable changes in endogenous IAA. Therefore, the reduction in IAA appears to result from an ABA-independent effect of NaCl on IAA metabolism in the roots of stressed plants.     

Pleiotropic effects of the<Emphasis Type="Italic"> male sterile33</Emphasis> (<Emphasis Type="Italic">ms33</Emphasis>) mutation in<Emphasis Type="Italic"> Arabidopsis</Emphasis> are associated with modifications in endogenous gibberellins,indole-3-acetic acid and abscisic acid

Earlier, we reported that mutation in the Male Sterile33 (MS33) locus in Arabidopsis thaliana causes inhibition of stamen filament growth and a defect in the maturation of pollen grains [Fei and Sawhney (1999) Physiol Plant 105:165–170; Fei and Sawhney (2001) Can J Bot 79:118–129]. Here we report that the ms33 mutant has other pleiotropic effects, including aberrant growth of all floral organs and a delay in seed germination and in flowering time. These defects could be partially or completely restored by low temperature or by exogenous gibberellin A₄ (GA₄), which in all cases was more effective than GA₃ Analysis of endogenous GAs showed that in wild type (WT) mature flowers GA₄ was the major GA, and that relative to WT the ms33 flowers had low levels of the growth active GAs, GA₁ and GA₄, and very reduced levels of GA₉, GA₂₄ and GA₁₅, precursors of GA₄. This suggests that mutation in the MS33 gene may suppress the GA biosynthetic pathway that leads to GA₄ via GA₉ and the early 13-H C₂₀ GAs. WT flowers also possessed a much higher level of indole-3-acetic acid (IAA), and a lower level of abscisic acid (ABA), relative to ms33 flowers. Low temperature induced partial restoration of male fertility in the ms33 flowers and this was associated with partial increase in GA₄. In contrast, in WT flowers GA₁ and GA₄ were very much reduced by low temperature. Low temperature also had little effect on IAA or ABA levels of ms33 flowers, but did reduce (>2-fold) IAA levels in WT flowers. The double mutants, ms33 aba1-1 (an ABA-deficient mutant), and ms33 spy-3 (a GA signal transduction mutant) had flower phenotypes similar to ms33. Together, the data suggest that the developmental defects in the ms33 mutant are unrelated to ABA levels, but may be causally associated with reduced levels of IAA, GA₁ and GA₄, compared to WT flowers.Abbreviations ABA Abscisic acid - GA Gibberellin - GC-MS-SIM Gas chromatography-mass spectrometry-selected ion monitoring - IAA Indole-3-acetic acid - ms33 Male sterile33 mutant - PP333 Paclobutrazol - WT Wild type