Expression of PaNAC01, a Picea abies CUP-SHAPED COTYLEDON orthologue, is regulated by polar auxin transport and associated with differentiation of the shoot apical meristem and formation of separated cotyledons

Background and Aims During embryo development in most gymnosperms, the establishment of the shoot apical meristem (SAM) occurs concomitantly with the formation of a crown of cotyledons surrounding the SAM. It has previously been shown that the differentiation of cotyledons in somatic embryos of Picea abies is dependent on polar auxin transport (PAT). In the angiosperm model plant, Arabidopsis thaliana, the establishment of cotyledonary boundaries and the embryonal SAM is dependent on PAT and the expression of the CUP-SHAPED COTYLEDON (CUC) genes, which belong to the large NAC gene family. The aim of this study was to characterize CUC-like genes in a gymnosperm, and to elucidate their expression during SAM and cotyledon differentiation, and in response to PAT. Methods Sixteen Picea glauca NAC sequences were identified in GenBank and deployed to different clades within the NAC gene family using maximum parsimony analysis and Bayesian inference. Motifs conserved between angiosperms and gymnosperms were analysed using the motif discovery tool MEME. Expression profiles during embryo development were produced using quantitative real-time PCR. Protein conservation was analysed by introducing a P. abies CUC orthologue into the A. thaliana cuc1cuc2 double mutant. Key Results Two full-length CUC-like cDNAs denoted PaNAC01 and PaNAC02 were cloned from P. abies. PaNAC01, but not PaNAC02, harbours previously characterized functional motifs in CUC1 and CUC2. The expression profile of PaNAC01 showed that the gene is PAT regulated and associated with SAM differentiation and cotyledon formation. Furthermore, PaNAC01 could functionally substitute for CUC2 in the A. thaliana cuc1cuc2 double mutant. Conclusions The results show that CUC-like genes with distinct signature motifs existed before the separation of angiosperms and gymnosperms approx. 300 million years ago, and suggest a conserved function between PaNAC01 and CUC1/CUC2.     

Auxin Polar Transport Is Essential for the Establishment of Bilateral Symmetry during Early Plant Embryogenesis

We used an in vitro culture system to investigate the effects of three auxin polar transport inhibitors (9-hydroxyfluorene-9-carboxylic acid, trans-cinnamic acid, and 2,3,5-triiodobenzoic acid) on the development of early globular to heart-shaped stage embryos of Indian mustard (Brassica juncea) plants. Although the effective concentrations vary with the different inhibitors, all of them induced the formation of fused cotyledons in globular ([less than or equal to]60 [mu]m) but not heart-shaped embryos. Inhibitor-treated Brassica embryos phenocopy embryos of the Arabidopsis pin-formed mutant pin1-1, which has reduced auxin polar transport activity in inflorescence axes, as well as embryos of the Arabidopsis emb30 (gnom) mutant. These results indicate that the polar transport of auxin in early globular embryos is essential for the establishment of bilateral symmetry during plant embryogenesis. Based on these observations, we propose two possible models for the action of auxin during cotyledon formation.     

The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport

Arabidopsis pinoid mutants show a strong phenotypic resemblance to the pin-formed mutant that is disrupted in polar auxin transport. The PINOID gene was recently cloned and found to encode a protein-serine/threonine kinase. Here we show that the PINOID gene is inducible by auxin and that the protein kinase is present in the primordia of cotyledons, leaves and floral organs and in vascular tissue in developing organs or proximal to meristems. Overexpression of PINOID under the control of the constitutive CaMV 35S promoter (35S::PID) resulted in phenotypes also observed in mutants with altered sensitivity to or transport of auxin. A remarkable characteristic of high expressing 35S::PID seedlings was a frequent collapse of the primary root meristem. This event triggered lateral root formation, a process that was initially inhibited in these seedlings. Both meristem organisation and growth of the primary root were rescued when seedlings were grown in the presence of polar auxin transport inhibitors, such as naphthylphtalamic acid (NPA). Moreover, ectopic expression of PINOID cDNA under control of the epidermis-specific LTP1 promoter provided further evidence for the NPA-sensitive action of PINOID. The results presented here indicate that PINOID functions as a positive regulator of polar auxin transport. We propose that PINOID is involved in the fine-tuning of polar auxin transport during organ formation in response to local auxin concentrations.     

Enhanced Polar Auxin Transport in Tomato Polycotyledon Mutant Seems to be Related to Glutathione Levels

Glutathione-dependent root growth in Arabidopsis is linked to polar auxin transport (PAT). Arabidopsis mutants with reduced glutathione (GSH) levels also show reduced PAT. To gain an insight into the relationship between PAT and GSH level, we analyzed tomato polycotyledon mutant, pct1-2, which has enhanced PAT. Microarray analysis of gene expression in pct1-2 mutant revealed underexpression of several genes related to glutamate and glutathione metabolism. In consonance with microarray analysis, enzymatic as well as in vivo assay revealed higher glutathione levels in the early phase of pct1-2 seedling growth than WT. The inhibition of auxin transport by 2,3,5-triiodobenzoic acid (TIBA) reduced both GSH level and PIN1 expression in pct1-2 root tips. The reduction of in vivo GSH accumulation in pct1-2 root tips by buthionine sulfoximine (BSO) stimulated elongation of the short root of pct1-2 mutant akin to TIBA. The rescue of the short root phenotype of the pct1-2 mutant was restricted to TIBA and BSO. The other auxin transport inhibitors 1-N-naphthylphthalamic acid (NPA), 2-[4-(diethylamino)-2-hydroxybenzoyl] benzoic acid (BUM), 3-chloro-4-hydroxyphenylacetic acid (CHPAA), brefeldin and gravacin inhibited root elongation in both WT and pct1-2 mutant. Our results indicate a relationship between PAT and GSH levels in tomato akin to Arabidopsis. Our work also highlights that TIBA rescues the short root phenotype of the pct1-2 mutant by acting on a PAT component distinct from the site of action of other PAT inhibitors.

     

Evaluating auxin distribution in tomato (Solanum lycopersicum) through an analysis of the PIN and AUX/LAX gene families

The temporal and spatial control of auxin distribution has a key role in the regulation of plant growth and development, and much has been learnt about the mechanisms that influence auxin pools and gradients in vegetative tissues, particularly in Arabidopsis. For example polar auxin transport, mediated by PIN and AUX/LAX proteins, is central to the control of auxin distribution. In contrast, very little information is known about the dynamics of auxin distribution and the molecular basis of its transport within and between fruit tissues, despite the fact that auxin regulates many aspects of fruit development, which include fruit formation, expansion, ripening and abscission. In addition, functional information regarding the key regulators of auxin fluxes during both vegetative and reproductive development in species other than Arabidopsis is scarce. To address these issues, we have investigated the spatiotemporal distribution of auxin during tomato (Solanum lycopersicum) fruit development and the function of the PIN and AUX/LAX gene families. Differential concentrations of auxin become apparent during early fruit growth, with auxin levels being higher in internal tissues than in the fruit pericarp and the pattern of auxin accumulation depended on polar transport. Ten tomato PIN (SlPIN1 to 10) and five AUX/LAX (SlLAX1 to 5) genes were identified and found to display heterogeneous expression patterns, with tissue and developmental-stage specificity. RNAi-mediated co-silencing of SlPIN4 and SlPIN3 did not affect fruit development, which suggested functional redundancy of PIN proteins, but did lead to a vegetative phenotype, and revealed a role for these genes in the regulation of tomato shoot architecture.     

SOS3 mediates lateral root development under low salt stress through regulation of auxin redistribution and maxima in Arabidopsis

? The SOS signaling pathway plays an important role in plant salt tolerance. However, little is known about how the SOS pathway modulates organ development in response to salt stress. Here, the involvement of SOS signaling in NaCl-induced lateral root (LR) development in Arabidopsis was assessed. ? Wild-type and sos3-1 mutant seedlings on iso-osmotic concentrations of NaCl and mannitol were analyzed. The marker lines for auxin accumulation, auxin transport, cell division activity and stem cells were also examined. ? The results showed that ionic effect alleviates the inhibitory effects of osmotic stress on LR development. LR development of the sos3-1 mutant showed increased sensitivity specifically to low salt. Under low-salt conditions, auxin in cotyledons and LR primordia (LRP) of the sos3-1 mutant was markedly reduced. Decreases in auxin polar transport of mutant roots may cause insufficient auxin supply, resulting in defects not only in LR initiation but also in cell division activity in LRP. ? Our data uncover a novel role of the SOS3 gene in modulation of LR developmental plasticity and adaptation in response to low salt stress, and reveal a new mechanism for plants to sense and adapt to small changes of salt.     

Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato

In this study we investigated the role of ethylene in the formation of lateral and adventitious roots in tomato ( Solanum lycopersicum ) using mutants isolated for altered ethylene signaling and fruit ripening. Mutations that block ethylene responses and delay ripening – Nr ( Never ripe ), gr ( green ripe ), nor ( non ripening ), and rin ( ripening inhibitor ) – have enhanced lateral root formation. In contrast, the epi ( epinastic ) mutant, which has elevated ethylene and constitutive ethylene signaling in some tissues, or treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC), reduces lateral root formation. Treatment with ACC inhibits the initiation and elongation of lateral roots, except in the Nr genotype. Root basipetal and acropetal indole-3-acetic acid (IAA) transport increase with ACC treatments or in the epi mutant, while in the Nr mutant there is less auxin transport than in the wild type and transport is insensitive to ACC. In contrast, the process of adventitious root formation shows the opposite response to ethylene, with ACC treatment and the epi mutation increasing adventitious root formation and the Nr mutation reducing the number of adventitious roots. In hypocotyls, ACC treatment negatively regulated IAA transport while the Nr mutant showed increased IAA transport in hypocotyls. Ethylene significantly reduces free IAA content in roots, but only subtly changes free IAA content in tomato hypocotyls. These results indicate a negative role for ethylene in lateral root formation and a positive role in adventitious root formation with modulation of auxin transport as a central point of ethylene–auxin crosstalk.     

Mitochondrial heat-shock cognate protein 70 contributes to auxin-mediated embryo development

In Arabidopsis thaliana, mitochondrial-localized heat-shock cognate protein 70-1 (mtHSC70-1) plays an important role in vegetativegrowth. However, whether mtHSC70-1 affects reproductive growth remains unknown. Here, we found that the mtHSC70-1 gene was expressed in the provascular cells of the embryo proper from the early heart stage onward during embryogenesis. Phenotypic analyses of mthsc70-1 mutants revealed that mtHSC70 deficiency leads to defective embryo development and that this effect is mediated by auxin. In addition to a dwarf phenotype, the mthsc70-1 mutant displayed defects in flower morphology, anther development, and embryogenesis. At early developmental stages, the mthsc70-1 embryos exhibited abnormal cell divisions in both embryo proper and suspensor cells. From heart stage onward, they displayed an abnormal shape such as with no or very small cotyledon protrusions, had aberrant number of cotyledons, or were twisted. These embryo defects were associated with reduced or ectopic expression of auxin responsive reporter DR5_rev:GFP. Consistently, the expression of auxin biosynthesis and polar auxin transport genes were markedly altered in mthsc70-1. On the other hand, mitochondrial retrograde regulation (MRR) was enhanced in mthsc70-1. Treatment of wild-type plants with an inhibitor that activates mitochondrial retrograde signaling reduced the expression level of auxin biosynthesis and polar auxin transport genes and induced phenotypes similar to those of mthsc70-1. Taken together, our data reveal that loss of function of mtHSC70-1 induces MRR, which inhibits auxin biosynthesis and polar auxin transport, leading to abnormal auxin gradients and defective embryo development.

mtHSC70-1 dysfunction induces mitochondrial retrograde regulation, which inhibits auxin biosynthesis and polar auxin transport, leading to abnormal auxin gradients and defective embryo development.     

Auxin transport into cotyledons and cotyledon growth depend similarly on the ABCB19 Multidrug Resistance-like transporter

ABCB19 of Arabidopsis thaliana (formerly known as MDR1 and PGP19) belongs to the Multidrug Resistance-like ( MDR ) or B group of the ATP-binding cassette (ABC) transporter superfamily, and mediates polar auxin transport in stems and roots. Here we have investigated the role of ABCB19 and auxin distribution in cotyledon development. During embryogenesis, confocal microscopy showed ABCB19 protein to be present in cotyledons during their main growth phase, but not later. Analysis of ProDR5:GFP expression patterns showed a significantly diminished and restricted auxin distribution pattern in abcb19 cotyledons. Nonetheless, development of abcb19 embryonic cotyledons was very similar to that of wild-type. Post-germination, ABCB19 was present in the plasma membrane of cotyledon epidermal, mesophyll and petiole cells during blade expansion. Post-germination cotyledon blade expansion in abcb19 was 65% slower than in wild-type, although the epidermal cell area was reduced by only 17%. The growth rate reduction quantitatively correlated with reduced auxin levels rather than auxin sensitivity as indicated by quantitative ProDR5:GUS assays and direct auxin measurements, and may be explained by the 50% reduction in the import of auxin through the petioles of abcb19 cotyledons during the period of maximum expansion. Taken together, these data indicate that cotyledon expansion during the establishment of photoautotrophic growth depends on ABCB19-mediated auxin import.     

CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants

Calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK) mediate plant responses to a variety of external stresses. Here we report that Arabidopsis CIPK6 is also required for the growth and development of plants. Phenotype of tobacco plants ectopically expressing a homologous gene ( CaCIPK6 ) from the leguminous plant chickpea ( Cicer arietinum ) indicated its functional conservation. A lesion in AtCIPK6 significantly reduced shoot-to-root and root basipetal auxin transport, and the plants exhibited developmental defects such as fused cotyledons, swollen hypocotyls and compromised lateral root formation, in conjunction with reduced expression of a number of genes involved in auxin transport and abiotic stress response. The Arabidopsis mutant was more sensitive to salt stress compared to wild-type, while overexpression of a constitutively active mutant of CaCIPK6 promoted salt tolerance in transgenic tobacco. Furthermore, tobacco seedlings expressing the constitutively active mutant of CaCIPK6 showed a developed root system, increased basipetal auxin transport and hypersensitivity to auxin. Our results provide evidence for involvement of a CIPK in auxin transport and consequently in root development, as well as in the salt-stress response, by regulating the expression of genes.     

Genetic evidence for auxin involvement in arbuscular mycorrhiza initiation

? Formation of arbuscular mycorrhiza (AM) is controlled by a host of small, diffusible signaling molecules, including phytohormones. To test the hypothesis that the plant hormone auxin controls mycorrhiza development, we assessed mycorrhiza formation in two mutants of tomato (Solanum lycopersicum): diageotropica (dgt), an auxin-resistant mutant, and polycotyledon (pct), a mutant with hyperactive polar auxin transport. ? Mutant and wild-type (WT) roots were inoculated with spores of the AM fungus Glomus intraradices. Presymbiotic root-fungus interactions were observed in root organ culture (ROC) and internal fungal colonization was quantified both in ROC and in intact seedlings. ? In ROC, G. intraradices stimulated presymbiotic root branching in pct but not in dgt roots. pct roots stimulated production of hyphal fans indicative of appressorium formation and were colonized more rapidly than WT roots. By contrast, approaching hyphae reversed direction to grow away from cultured dgt roots and failed to colonize them. In intact seedlings, pct and dgt roots were colonized poorly, but development of hyphae, arbuscules, and vesicles was morphologically normal within roots of both mutants. ? We conclude that auxin signaling within host roots is required for the early stages of AM formation, including during presymbiotic signal exchange.     

SEMAPHORE1 functions during the regulation of ancestrally duplicated knox genes and polar auxin transport in maize

The expression of class 1 knotted1-like homeobox (knox) genes affects numerous plant developmental processes, including cell-fate acquisition, lateral organ initiation, and maintenance of shoot apical meristems. The SEMAPHORE1 gene product is required for the negative regulation of a subset of maize knox genes, the duplicated loci rough sheath 1 and gnarley1 (knox4). Recessive mutations in semaphore1 result in the ectopic expression of knox genes in leaf and endosperm tissue. Genetic analyses suggest that SEMAPHORE1 may regulate knox gene expression in a different developmental pathway than ROUGH SHEATH2, the first-identified regulator of knox gene expression in maize. Mutations at semaphore1 are pleiotropic, disrupting specific domains of the shoot. However, unlike previously described mutations that cause ectopic knox gene expression, semaphore1 mutations affect development of the embryo, endosperm, lateral roots, and pollen. Moreover, polar transport of the phytohormone auxin is significantly reduced in semaphore1 mutant shoots. The data suggest that many of the pleiotropic semaphore1 phenotypes result from defective polar auxin transport (PAT) in sem1 mutant shoots, and support models correlating down-regulated knox gene expression and PAT in maize shoots.     

Alteration of auxin polar transport in the Arabidopsis ifl1 mutants

The INTERFASCICULAR FIBERLESS/REVOLUTA (IFL1/REV) gene is essential for the normal differentiation of interfascicular fibers and secondary xylem in the inflorescence stems of Arabidopsis. It has been proposed that IFL1/REV influences auxin polar flow or the transduction of auxin signal, which is required for fiber and vascular differentiation. Assay of auxin polar transport showed that the ifl1 mutations dramatically reduced auxin polar flow along the inflorescence stems and in the hypocotyls. The null mutant allele ifl1-2 was accompanied by a significant decrease in the expression level of two putative auxin efflux carriers. The ifl1 mutants remained sensitive to auxin and an auxin transport inhibitor. The ifl1-2 mutant exhibited visible phenotypes associated with defects in auxin polar transport such as pin-like inflorescence, reduced numbers of cauline branches, reduced numbers of secondary rosette inflorescence, and dark green leaves with delayed senescence. The visible phenotypes displayed by the ifl1 mutants could be mimicked by treatment of wild-type plants with an auxin polar transport inhibitor. In addition, the auxin polar transport inhibitor altered the normal differentiation of interfascicular fibers in the inflorescence stems of wild-type Arabidopsis. Taken together, these results suggest a correlation between the reduced auxin polar transport and the alteration of cell differentiation and morphology in the ifl1 mutants.     

生长素极性运输在水稻根发育中的作用

生长素极性运输(PAT)在植物生长发育尤其是极性发育和模式建成中起重要作用.采用2种PAT抑制剂TIBA(2,3,5-triiodobenzoic acid)和HFCA(9-hydroxyfluorene-9-carboxylic acid)处理水稻(Oryza sativa L. cv.Zhonghua)幼苗,结果表明:PAT影响水稻根发育包括主根的伸长、侧根的起始和伸长以及不定根的发育.PAT的抑制导致主根变短、侧根和不定根数目减少.外源附加生长素(NAA)可以部分恢复不定根的形成但不能恢复侧根的形成,表明在侧根和不定根的形成上可能具有不同的机制.切片结果表明,30μmol/TIBA处理后并不完全抑制侧根原基的形成,进一步研究表明生长素由胚芽鞘向基部的运输在水稻不定根的起始和伸长中起关键作用.     

Auxin Depletion from the Leaf Axil Conditions Competence for Axillary Meristem Formation in Arabidopsis and Tomato

The enormous variation in architecture of flowering plants is based to a large extent on their ability to form new axes of growth throughout their life span. Secondary growth is initiated from groups of pluripotent cells, called meristems, which are established in the axils of leaves. Such meristems form lateral organs and develop into a side shoot or a flower, depending on the developmental status of the plant and environmental conditions. The phytohormone auxin is well known to play an important role in inhibiting the outgrowth of axillary buds, a phenomenon known as apical dominance. However, the role of auxin in the process of axillary meristem formation is largely unknown. In this study, we show in the model species Arabidopsis thaliana and tomato (Solanum lycopersicum) that auxin is depleted from leaf axils during vegetative development. Disruption of polar auxin transport compromises auxin depletion from the leaf axil and axillary meristem initiation. Ectopic auxin biosynthesis in leaf axils interferes with axillary meristem formation, whereas repression of auxin signaling in polar auxin transport mutants can largely rescue their branching defects. These results strongly suggest that depletion of auxin from leaf axils is a prerequisite for axillary meristem formation during vegetative development.