H<Subscript>2</Subscript>O<Subscript>2</Subscript> regulates root system architecture by modulating the polar transport and redistribution of auxin

The accumulation and redistribution of the plant hormone auxin plays a crucial role in root development and patterning. Plants can alter their root system architecture (RSA) to adapt to different biotic and abiotic stresses. In addition, reactive oxygen species (ROS), such as H₂O₂, are known to increase in plants undergoing stress. Here, we present evidence that H₂O₂ can regulate auxin accumulation and redistribution through modulating polar auxin transport, leading to changes in RSA. Plants exposed to different concentrations of H₂O₂ formed a highly branched root system with abundant lateral roots and a shorter primary root. Monitoring of the auxin responsive DR5::GUS indicated that auxin accumulation decreased in lateral root primordia (LRP) and emerging lateral root tips. In addition, polar auxin transport, including both basipetal and acropetal transport modulated by AUX1 and PIN protein carriers, was involved in the process. Taken together, our results suggest that H₂O₂ could regulate plastic RSA by perturbing polar auxin transport as a means of modulating the accumulation and distribution of auxin.     

Activation of a flavin monooxygenase gene <Emphasis Type="Italic">YUCCA7</Emphasis> enhances drought resistance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin regulates diverse molecular and physiological events at the cellular and organismal levels during plant growth and development in response to environmental stimuli. It acts either through distinct signaling pathways or in concert with other growth hormones. Its biological functions are adjusted by modulating biosynthesis, conjugate formation, and polar transport and distribution. Several tryptophan-dependent and -independent auxin biosynthetic pathways have been proposed. Recent studies have shown that a few flavin monooxygenase enzymes contribute to the tryptophan-dependent auxin biosynthesis. Here, we show that activation of a flavin monooxygenase gene YUCCA7 (YUC7), which belongs to the tryptophan-dependent auxin biosynthetic pathway, enhances drought resistance. An Arabidopsis activation-tagged mutant yuc7-1D exhibited phenotypic changes similar to those observed in auxin-overproducing mutants, such as tall, slender stems and curled, narrow leaves. Accordingly, endogenous levels of total auxin were elevated in the mutant. The YUC7 gene was induced by drought, primarily in the roots, in an abscisic acid (ABA)-dependent manner. The yuc7-1D mutant was resistant to drought, and drought-responsive genes, such as RESPONSIVE TO DESSICATION 29A (RD29A) and COLD-REGULATED 15A (COR15A), were up-regulated in the mutant. Interestingly, whereas stomatal aperture and production of osmoprotectants were not discernibly altered, lateral root growth was significantly promoted in the yuc7-1D mutant when grown under drought conditions. These observations support that elevation of auxin levels in the roots enhances drought resistance possibly by promoting root growth.     

Localized auxin biosynthesis and postembryonic root development in Arabidopsis

Auxin is a phytohormone essential for plant development. Due to the high redundancy in auxin biosynthesis, the role of auxin biosynthesis in embryogenesis and seedling development, vascular and flower development, shade avoidance and ethylene response were revealed only recently. We previously reported that a vitamin B₆ biosynthesis mutant pdx1 exhibits a short-root phenotype with reduced meristematic zone and short mature cells. By reciprocal grafting, we now have found that the pdx1 short root is caused by a root locally generated signal. The mutant root tips are defective in callus induction and have reduced DR5::GUS activity, but maintain relatively normal auxin response. Genetic analysis indicates that pdx1 mutant could suppress the root hair and root growth phenotypes of the auxin overproduction mutant yucca on medium supplemented with tryptophan (Trp), suggesting that the conversion from Trp to auxin is impaired in pdx1 roots. Here we present data showing that pdx1 mutant is more tolerant to 5-methyl anthranilate, an analogue of the Trp biosynthetic intermediate anthranilate, demonstrating that pdx1 is also defective in the conversion from anthranilate to auxin precursor tryptophan. Our data suggest that locally synthesized auxin may play an important role in the postembryonic root growth.Key words: auxin synthesis, root, PLP, PDX1The plant hormone auxin modulates many aspects of growth and development including cell division and cell expansion, leaf initiation, root development, embryo and fruit development, pattern formation, tropism, apical dominance and vascular tissue differentiation.^1–3 Indole-3-acetic acid (IAA) is the major naturally occurring auxin. IAA can be synthesized in cotyledons, leaves and roots, with young developing leaves having the highest capacity.^4,5Auxin most often acts in tissues or cells remote from its synthetic sites, and thus depends on non-polar phloem transport as well as a highly regulated intercellular polar transport system for its distribution.²The importance of local auxin biosynthesis in plant growth and development has been masked by observations that impaired long-distance auxin transport can result in severe growth or developmental defects.^3,6 Furthermore, a few mutants with reduced free IAA contents display phenotypes similar to those caused by impaired long-distance auxin transport. These phenotypes include defective vascular tissues and flower development, short primary roots and reduced apical dominance, or impaired shade avoidance and ethylene response.^7–15 Since these phenotypes most often could not be rescued by exogenous auxin application, it is difficult to attribute such defects to altered local auxin biosynthesis. By complementing double, triple or quadruple mutants of four Arabidopsis shoot-abundant auxin biosynthesis YUCCA genes with specific YUCCA promoters driven bacterial auxin biosynthesis iaaM gene, Cheng et al. provided unambiguous evidence that auxin biosynthesis is indispensable for embryo, flower and vascular tissue development.^8,13 Importantly, it is clear that auxin synthesized by YUCCAs is not functionally interchangeable among different organs, supporting the notion that auxin synthesized by YUCCAs mainly functions locally or in a short range.^6,8,13The central role of auxin in root meristem patterning and maintenance is well documented,^1,2,16 but the source of such IAA is still unclear. When ¹⁴C-labeled IAA was applied to the five-day-old pea apical bud, the radioactivity could be detected in lateral root primordia but not the apical region of primary roots.¹⁷ Moreover, removal of the shoot only slightly affected elongation of the primary root, and localized application of auxin polar transport inhibitor naphthylphthalamic acid (NPA) at the primary root tip exerted more profound inhibitory effect on root elongation than at any other site.¹⁸ These results suggest that auxin generated near the root tip may play a more important role in primary root growth than that transported from the shoot. In line with this notion, Arabidopsis roots have been shown to harbor multiple auxin biosynthesis sites including root tips and the region upward from the tip.⁴Many steps of tryptophan synthesis and its conversion to auxin involve transamination reactions, which require the vitamin B₆ pyridoxal 5-phosphate (PLP) as a cofactor. We previously reported that the Arabidopsis mutant pdx1 that is defective in vitamin B₆ biosynthesis displays dramatically reduced primary root growth with smaller meristematic zone and shorter mature cortical cells.¹⁹ In the current investigation, we found that the root tips of pdx1 have reduced cell division capability and reduced DR5::GUS activity, although the induction of this reporter gene by exogenous auxin was not changed. Reciprocal grafting indicates that the short-root phenotype of pdx1 is caused by a root local rather than shoot generated factor(s). Importantly, pdx1 suppresses yucca mutant, an auxin overproducer, in root hair proliferation although it fails to suppress the hypocotyl elongation phenotype.²⁰ Our work thus demonstrated that pdx1 has impaired root local auxin biosynthesis from tryptophan. To test whether the synthesis of tryptophan is also affected in pdx1 mutant, we planted pdx1 together with wild-type seeds on Murashige and Skoog (MS) medium supplemented with 5-mehtyl-anthranilate (5-MA), an analogue of the Trp biosynthetic intermediate anthranilate.²¹ Although pdx1 seedlings grew poorly under the control conditions, the growth of wild-type seedlings was more inhibited than that of the pdx1 seedlings on 10 µM 5-MA media (Fig. 1A–D). Compared with the elongated primary root on MS, wild-type seedlings showed very limited root growth on 5-MA (Fig. 1E). The relatively increased tolerance to 5-MA of pdx1 thus indicates that the pdx1 mutant may be defective in Trp biosynthesis, although amino acid analysis of the bulked seedlings did not find clear changes in Trp levels in the mutants (our unpublished data).Open in a separate windowFigure 1The pdx1 mutant seedlings are relatively less sensitive to toxic 5-methyl anthranilate (5-MA). (A and C) Five-day-old seedlings of the wild type (Col-0) (A) or pdx1 (C) on MS medium. (B and D) Five-day-old seedlings of the wild type (B) or pdx1 (D) on MS medium supplemented with 10 µM 5-MA. (E) Eight-day-old seedlings of the wild type or pdx1 on MS medium without or with 10 µM 5-MA supplement. Sterilized seeds were planted directly on the indicated medium and after two days of cold treatment, the plates were incubated under continuous light at 22–24°C before taking pictures.We reported that PDX1 is required for tolerance to oxidative stresses in Arabidopsis.¹⁹ Interestingly, redox homeostasis appears to play a critical role in Arabidopsis root development. The glutathione-deficient mutant root meristemless1 (rml1) and the vitamin C-deficient mutant vitamin C1 (vtc1) both have similar stunted roots.^22,23 Nonetheless, pdx1 is not rescued by either glutathione or vitamin C¹⁹ suggesting that the pdx1 short-root phenotype may not be resulted from a general reduction of antioxidative capacity. Interestingly, ascorbate oxidase is found to be highly expressed in the maize root quiescent center.²⁴ This enzyme can oxidatively decarboxylate auxin in vitro, suggesting that the quiescent center may be a site for metabolizing auxin to control its homeostasis.²⁵ It is therefore likely that the reduced auxin level in pdx1 root tips could be partially caused by increased auxin catabolism resulted from reduced vitamin B₆ level. We thus conducted experiments to test this possibility. A quiescent center-specific promoter WOX5 driven bacterial auxin biosynthetic gene iaaH²⁶ was introduced into pdx1 mutant. The transgenic seeds were planted on media supplemented with different concentrations of indoleacetamide (IAM), the substrate of iaaH protein. Although promotion of lateral root growth was observed at higher IAM concentrations, which indicates increased tryptophan-independent auxin production from the transgene, no change in root elongation was observed between pdx1 with or without the WOX5::iaaH transgene at any concentration of IAM tested (data not shown), suggesting that the pdx1 short-root phenotype may not be due to increased auxin catabolism.Taken together, in addition to auxin transport; temporally, spatially or developmentally coordinated local auxin biosynthesis defines the plant growth and its response to environmental changes.^8,14,15     

High Temperature Stimulates <Emphasis Type="Italic">DWARF4 (DWF4)</Emphasis> Expression to Increase Hypocotyl Elongation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Hypocotyl growth occurs as a result of an interaction between environmental factors and endogenous phytohormones. In Arabidopsis, high temperature promotes auxin synthesis to increase hypocotyl growth. We previously showed that exogenously provided auxin stimulates expression of the brassinosteroid (BR) biosynthetic gene DWARF4. To determine whether temperature-induced hypocotyl elongation depends on BR biosynthesis, we examined the morphological responses to high temperature and the expression pattern of DWF4pro:GUS in different genetic backgrounds, which are as follows: Ws-2 wild-type, iaa19/msg2, bri1-5, and dwf7-1. In contrast to the wild-type, growth of the three genotypes at 29°C did not significantly increase hypocotyl length; whereas, with the exception of iaa19/msg2, the roots were elongated. These results confirm that BR biosynthesis and signaling pathways are required for hypocotyl growth at high temperature. Furthermore, a GUS histochemical assay revealed that a temperature of 29°C greatly increased DWF4pro:GUS expression in the shoot and root tips compared to a temperature of 22°C. Quantitative measurements of GUS activity in DWF4pro:GUS revealed that growth at 29°C is similar to the level of growth after addition of 100 nM IAA to the medium. Our results suggest that temperature-dependent synthesis of free auxin stimulates BR biosynthesis, particularly via the key biosynthetic gene DWF4, and that the BRs thus synthesized are involved in hypocotyl growth at high temperature.     

OsPIN2, which encodes a member of the auxin efflux carrier proteins,is involved in root elongation growth and lateral root formation patterns via the regulation of auxin distribution in rice

Auxin flow is important for different root developmental processes such as root formation, emergence, elongation and gravitropism. However, the detailed information about the mechanisms regulating the auxin flow is less well understood in rice. We characterized the auxin transport‐related mutants, Ospin‐formed2‐1 (Ospin2‐1) and Ospin2‐2, which exhibited curly root phenotypes and altered lateral root formation patterns in rice. The OsPIN2 gene encodes a member of the auxin efflux carrier proteins that possibly regulates the basipetal auxin flow from the root tip toward the root elongation zone. According to DR5‐driven GUS expression, there is an asymmetric auxin distribution in the mutants that corresponded with the asymmetric cell elongation pattern in the mutant root tip. Auxin transport inhibitor, N‐1‐naphthylphthalamic acid and Ospin2‐1 Osiaa13 double mutant rescued the curly root phenotype indicating that this phenotype results from a defect in proper auxin distribution. The typical curly root phenotype was not observed when Ospin2‐1 was grown in distilled water as an alternative to tap water, although higher auxin levels were found at the root tip region of the mutant than that of the wild‐type. Therefore, the lateral root formation zone in the mutant was shifted basipetally compared with the wild‐type. These results reflect that an altered auxin flow in the root tip region is responsible for root elongation growth and lateral root formation patterns in rice.     

Titanium Dioxide Nanoparticles Promote Root Growth by Interfering with Auxin Pathways in <i>Arabidopsis thaliana</i>

TiO₂ nanoparticles (nano-TiO₂) are widely used in the world, and a considerable amount of nano-TiO₂ is released into the environment, with toxic effects on organisms. In the various species of higher plants, growth, including seed germination, root elongation, and biomass accumulation, is affected by nano-TiO₂. However, the underlying molecular mechanisms remain to be elucidated. In this study, we observed that nano-TiO₂ promoted root elongation in a dose-dependent manner. Furthermore, we found that nano-TiO₂ elevated auxin accumulation in the root tips of the auxin marker lines DII-VENUS and DR5:: GUS, and, correspondingly, quantitative real-time PCR analysis revealed that nano-TiO₂ increased the expression levels of auxin biosynthesis- and transportrelated genes. GFP fluorescence observation using transgenic PIN2-GFP indicated that nano-TiO₂ promoted root growth by inducing PIN2 accumulation. Thus, we propose that nano-TiO₂ promote root growth in Arabidopsis thaliana by altering the expression levels of auxin biosynthesis- and transport-related genes.     

BIN2/DWF12 Antagonistically Transduces Brassinosteroid and Auxin Signals in the Roots of <Emphasis Type="Italic">Arabidopsis</Emphasis>

Plant growth-stimulating hormones brassinosteroids (BRs) function via interactions with other hormones. However, the mechanism of these interactions remains to be elucidated. The unique phenotypes of brassinosteroid insensitive2/dwarf12-D (bin2/dwf12-D) mutants, such as twisted inflorescences and leaves, suggested that BIN2, a negative regulator of BR signaling, may be involved in auxin signaling. Furthermore, previously, we showed that auxin stimulates DWF4 expression. To determine the possible role of BIN2/DWF12 in Auxin signaling, we measured DWARF4pro:GUS activity through both GUS histochemical staining and in vivo GUS assay. We found that the GUS activity in the bin2/dwarf12-1D background dramatically increased relative to control. In addition, the number of lateral roots (LR) in bin2/dwf12-1D was greater than wild type, and the optimal concentration for auxin-mediated lateral root induction was lower in bin2/dwf12-1D; these findings suggest that BIN2 plays a positive role in auxin signaling. In contrast, ABA repressed both DWF4pro:GUS expression and lateral root development. However, the degree of repression was lower in bin2/dwf12-1D background, suggesting that BIN2 plays a role in ABA-mediated DWF4pro:GUS expression and subsequently in lateral root development, too. Therefore, it is likely that BIN2 plays a role of signal integrator for multiple hormones, such as BRs, auxin, and ABA.     

Analysis the role of arabidopsis <Emphasis Type="Italic">CKRC6/ASA1</Emphasis> in auxin and cytokinin biosynthesis

The crosstalk between auxin and cytokinin (CK) is important for plant growth and development, although the underlying molecular mechanisms remain unclear. Here, we describe the isolation and characterization of a mutant of Arabidopsis Cytokinin-induced Root Curling 6 (CKRC6), an allele of ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) that encodes the á-subunit of AS in tryptophan (Trp) biosynthesis. The ckrc6 mutant exhibits root gravitropic defects and insensitivity to both CK and the ethylene precursor 1-aminocyclopropane-1-carboxylicacid (ACC) in primary root growth. These defects can be rescued by exogenous indole-3-acetic acid (IAA) or tryptophan (Trp) supplementation. Furthermore, our results suggest that the ckrc6 mutant has decreased IAA content, differential expression patterns of auxin biosynthesis genes and CK biosynthesis isopentenyl transferase (IPT) genes in comparison to wild type. Collectively, our study shows that auxin controls CK biosynthesis based on that CK sensitivity is altered in most auxin-resistant mutants and that CKs promote auxin biosynthesis but inhibit auxin transport and response. Our results also suggest that CKRC6/ASA1 may be located at an intersection of auxin, CK and ethylene metabolism and/or signaling.     

BREVIS RADIX is involved in cytokinin-mediated inhibition of lateral root initiation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In contrast to auxin, relatively little is known about the molecular mechanism of cytokinin (CTK) inhibition of lateral root initiation. Previous studies demonstrated that BREVIS RADIX (BRX), a protein of unknown biochemical function, maintains a rate-limiting brassinosteroid biosynthesis enzyme expression to keep brassinosteroid biosynthesis above a critical threshold. Here, we show that the brx-2 mutant is insensitive to exogenous CTK-induced inhibition of lateral root initiation and that this can be restored by embryonic brassinosteroid treatment. However post-embryonic brassinosteroid treatment can not rescue brx-2 mutant phenotype in the presence of CTK. Meanwhile the brassinosteroid receptor defective mutant bri1-6 shows normal CTK-mediated inhibition on LR growth. These results suggest the CTK-mediated inhibition of LR initiation is not directly dependent on brassinosteroid level. Furthermore, compared with wild type, brx-2 exhibits altered auxin response in presumptive founder cells, lateral root primodia and primary root tip in the presence of exogenous CTK. We concluded that CTK inhibition on lateral root initiation depend on specific auxin response loss in presumptive founder cell. The aberrant primary root growth caused by the embryonic brassinosteroid shortage can indirectly result in the lateral root phenotype of brx-2 in presence of CTK. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A negative regulatory role for auxin in sulphate deficiency response in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Sulphate is a major macronutrient required for the synthesis of the sulphur (S)-containing amino acid cysteine and thus is critical for cellular metabolism, growth and development and response to various abiotic and biotic stresses. A recent genome-wide expression study suggested that several auxin-inducible genes were up-regulated by S deficiency in Arabidopsis. Here, we examined the relationship between auxin signaling and S deficiency. Investigation of DR5::GUS expression patterns indicates that auxin accumulation and/or response is suppressed by S deficiency. Consistently, S deficiency resulted in the suppression of lateral root development, but the axr1-3 mutant was insensitive to this response. Furthermore, the activation of the promoter for the putative thioglucosidase gene (At2g44460) by S deficiency was suppressed by auxin, cytokinin and abscisic acid (ABA). Interestingly, the activation of At2g44460 by S deficiency is regulated by the availability of carbon and nitrogen nutrients in a tissue-specific manner. These results demonstrate that auxin plays a negative role in signaling to S deficiency. Given that activation of the genes encoding the sulphate transporter SULTR1;2 and 5′-adenylylsulphate reductase APR2 are suppressed by cytokinin only, we hypothesize that while cytokinin may play an important role in general S deficiency response, auxin might be only involved in a subset of S deficiency responses such as the release of thiol groups from the S storage sources. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Developmental consequences of the <Emphasis Type="Italic">tumorous shoot development1</Emphasis> mutation,a novel allele of the cellulose-synthesizing <Emphasis Type="Italic">KORRIGAN1</Emphasis> gene

This work describes the further characterization of the tumorous shoot development1 (tsd1) mutant of Arabidopsis thaliana, which develops disorganized tumorous-like shoot tissue instead of organized leaves and stems. Map-based cloning revealed that tsd1 is a novel strong allele of the KOR1 gene, encoding a membrane-bound endo-1,4-β-d-glucanase involved in cellulose synthesis. To study developmental changes accompanying the aberrant growth of the tsd1 mutant, patterning in the meristems and the hormonal status were analysed by marker genes. Expression of key regulators of meristem maintenance, the CLV3 and STM genes, indicated the presence of numerous meristems in the tsd1 shoot callus. Expression of the LFY::GUS marker supported the ability of the tsd1 callus to form organ primordia, which however failed to develop further. An epidermal marker showed that the L1 layer was maintained only in distinct areas of the tsd1 callus, which could be a reason of disorganized shoot growth. In the tsd1 root meristem, quiescent center activity was lost early after germination, which caused differentiation of the root meristem. The spatial expression of genes reporting the auxin and cytokinin status was altered in the tsd1 mutant. Modifying the endogenous levels of these hormones partially rescued shoot and root development of the tsd1 mutant. Together, the work shows that TSD1/KOR1 is required for maintaining a correct meristematic pattern and organ growth as well as for a normal hormonal response.     

Influence of Zn<Superscript>2+</Superscript>, Co<Superscript>2+</Superscript> and dimethylbenzimidazole on vitamin B<Subscript>12</Subscript> biosynthesis by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis>

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B₁₂ biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn²⁺, Co²⁺ and DMBI on vitamin B₁₂ production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B₁₂. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B₁₂ production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.     

Probing the hormonal activity of fractionated molecular humic components in tomato auxin mutants

We progressively reduced the complexity of humic matter by a mild sequential removal of unbound or free components, weakly, and strongly bound molecules. The auxin‐like response of residues from each step was tested using tomato (cv. Micro‐Tom) seedlings expressing DR5 auxin synthetic promoter fused to the β‐glucuronidase (GUS) reporter gene and the low auxin‐sensitivity diageotropica (dgt) mutant. Both exogenous auxin and humic matter promoted lateral root emergence in the control, but failed to induce lateral roots in the dgt mutant. When strongly bound components were removed from humic matter by breaking the ester and ether bonds, the humic residues lost their ability to induce the DR5::GUS activity and lateral root emergence. However, these capacities were retained in the free or weakly bound molecules. These findings confirm that auxin‐like activity in humic matter is associated with complex hydrophobic structures, whose simplification by hydrolysis may release auxin‐like molecules.     

Identification of a Novel <i>OsCYP2</i> Allele that Was Involved in Rice Response to Low Temperature Stress

Cyclophilin (CYP) plays an important role in plant response to stress, and OsCYP2, one gene of cyclophlilin family, is involved in auxin signal transduction and stress signaling in rice. However, the mechanism that OsCYP2 is involved in rice response to low temperature is still unclear. We identified a new OsCYP2 allelic mutant, lrl3, with fewer lateral roots, and the differences in shoot height, primary root length and adventitious root length increased with the growth process compared to the wild-type plant. Auxin signaling pathway was also affected and became insensitive to gravity. The transgenic rice plants with over-expression of OsCYP2 were more tolerant to low temperature than the wild-type plants, suggesting that OsCYP2 was involved in the low temperature response in rice. In addition, OsCYP2 negatively regulated the expression of OsTPS38, a terpene synthase gene, and was dependent on the OsCDPK7-mediated pathway in response to low temperature stress. OsTPS38- overexpressed transgenic line ox-2 was more sensitive to low temperature. Therefore, OsCYP2 may negatively regulate OsTPS38 through an OsCDPK7-dependent pathway to mediate the response to low temperature in rice. These results provide a new basis for auxin signaling genes to regulate rice response to low temperature stress.     

Molecular expression of <Emphasis Type="Italic">PsPIN1</Emphasis>, a putative auxin efflux carrier gene from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

A cDNA coding for a putative auxin efflux carrier was amplified from Pisum sativum seedling shoot tips by RT-PCR and the corresponding full-length cDNA, PsPIN1, was subsequently obtained by RACE-PCR. The deduced amino acid sequence (599 residues) showed the three domain topology typical of the other PIN proteins. The PsPIN1 protein structure prediction possessed five transmembrane domains at both the N-(7-150) and C-(450-575) termini and a hydrophylic region in the middle. PsPIN1 showed highest similarity to Medicago, MtPIN4. Using the Genome Walking technique, a 1511 bp upstream region for PsPIN1 gene was sequenced. This PsPIN1 upstream region possessed multiple putative auxin, GA and light regulatory elements. The PsPIN1 mRNA was ubiquitously expressed throughout the pea plant, especially in growing tissues. Auxin induced PsPIN1 mRNA in dark grown pea seedling shoot tips. It was induced by 4-chloro-IAA, which is also an active auxin in pea, and by gibberellin (GA₃). Interestingly, the PsPIN1 mRNA was down-regulated by light treatment, possibly because light negatively regulates auxin and, especially GA levels in pea. Thus PIN1-mediated auxin efflux is a highly regulated process, not only at the level of protein localization, but also at the level of mRNA accumulation.     

The analysis of auxin distribution in the wild type and <Emphasis Type="Italic">abruptus</Emphasis> mutant plants of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) heynh. By using the chimeric gene <Emphasis Type="Italic">DR5::GUS</Emphasis>

We have studied the activity of the chimeric gene DR5::GUS that indicates the spatial pattern of free auxin distribution and relative auxin level in the tissues of A. thaliana plants. It is established that the temperature sensitive abruptus mutation leads to temperature dependent increased accumulation of the free auxin in leaves, staments, and sepals in comparison to control plants homozygous for the wild type allele ABRUPTUS/PINOID. Our data revealed the important role of the ABRUPTUS/PINOID gene in the regulation of the auxin polar transport.     

The Endosperm-Specific Expression of <i>YUCCA</i> Genes Enhances Rice Grain Filling

Grain filling is a crucial process that affects yield in rice (Oryza sativa L.). Auxin biosynthesis and signaling are closely related to rice yield; therefore, it is important to understand the effects of auxin biosynthesis on rice grain filling to improve crop yield. In this study, we used physiological and molecular strategies to identify the roles of auxin in rice grain filling. Exogenous application of auxin (IAA) or auxin analogues (2, 4-D) to young spikelets and flag leaves improved the seed-setting rate and yield per spike. Furthermore, real-time quantitative PCR assays confirmed that nine members of the OsYUCCA family of auxin biosynthetic genes were upregulated during grain filling, implication that auxin biosynthesis plays a major role in grain development. The specific expression of either Arabidopsis AtYUCCA1 or OsYUCCA2 in the endosperm or leaves resulted in increased expression of OsIAA genes and auxin content of seeds, as well as increased grain filling and seed-setting rate. This result establishes that the auxin content in grains and leaves is important for grain development. Our findings further highlight the potential applications for improving rice yield by elevating targeted gene expression in specific tissues.