PLANT PHYSIOLOGY DEVELOPMENT AND HORMONE ACTION

LBD18/ASL20 Regulates Lateral Root Formation in Combination with LBD16/ASL18 Downstream of ARF7 and ARF19 in Arabidopsis

Lee, H. W., Kim, N. Y., Lee, D. J., Kim, J. — Tue, 03 Nov 2009 12:22:27 PST

The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes encode proteins harboring a conserved amino acid domain, referred to as the LOB (for lateral organ boundaries) domain. While recent studies have revealed developmental functions of some LBD genes in Arabidopsis (Arabidopsis thaliana) and in crop plants, the biological functions of many other LBD genes remain to be determined. In this study, we have demonstrated that the lbd18 mutant evidenced a reduced number of lateral roots and that lbd16 lbd18 double mutants exhibited a dramatic reduction in the number of lateral roots compared with lbd16 or lbd18. Consistent with this observation, significant β-glucuronidase (GUS) expression in Pro_LBD18:GUS seedlings was detected in lateral root primordia as well as in the emerged lateral roots. Whereas the numbers of primordia of lbd16, lbd18, and lbd16 lbd18 mutants were similar to those observed in the wild type, the numbers of emerged lateral roots of lbd16 and lbd18 single mutants were reduced significantly. lbd16 lbd18 double mutants exhibited additively reduced numbers of emerged lateral roots compared with single mutants. This finding indicates that LBD16 and LBD18 may function in the initiation and emergence of lateral root formation via a different pathway. LBD18 was shown to be localized into the nucleus. We determined whether LBD18 functions in the nucleus using a steroid regulator-inducible system in which the nuclear translocation of LBD18 can be regulated by dexamethasone in the wild-type, lbd18, and lbd16 lbd18 backgrounds. Whereas LBD18 overexpression in the wild-type background induced lateral root formation to some degree, other lines manifested the growth-inhibition phenotype. However, LBD18 overexpression rescued lateral root formation in lbd18 and lbd16 lbd18 mutants without inducing any other phenotypes. Furthermore, we demonstrated that LBD18 overexpression can stimulate lateral root formation in auxin response factor7/19 (arf7 arf19) mutants with blocked lateral root formation. Taken together, our results suggest that LBD18 functions in the initiation and emergence of lateral roots, in conjunction with LBD16, downstream of ARF7 and ARF19.

Expressing the Diphtheria Toxin A Subunit from the HAP2(GCS1) Promoter Blocks Sperm Maturation and Produces Single Sperm-Like Cells Capable of Fertilization

Frank, A. C., Johnson, M. A. — Tue, 03 Nov 2009 12:22:27 PST

After meiosis, the male germline of flowering plants undergoes two mitoses, producing two sperm that are carried within a pollen tube to an ovule. One sperm fuses with the egg to form the zygote and the other fuses with the central cell to form the primary endosperm. The mechanisms that control male germline development and gene expression, and ensure that sperm properly fuse with female gametes are just beginning to be understood. Expression of the potent translation inhibitor, diphtheria toxin A subunit, from the Arabidopsis (Arabidopsis thaliana) HAP2(GCS1) promoter blocked sperm development before the final cell division, resulting in pollen tubes that carried a single sperm-like cell rather than two sperm. These pollen tubes targeted ovules and fertilized either the egg or the central cell, producing seeds with either endosperm or an embryo, but not both. Endosperm-only seeds significantly outnumbered embryo-only seeds, suggesting that single sperm-like cells preferentially fuse with the central cell. These experiments show that de novo translation is required for completion of sperm development, that the HAP2(GCS1) promoter is very tightly controlled, and that disruption of gene expression can result in male germ cells with a bias for gamete fusion.

The SPOROCYTELESS/NOZZLE Gene Is Involved in Controlling Stamen Identity in Arabidopsis

Tue, 03 Nov 2009 12:22:27 PST

The stamen, which consists of an anther and a filament, is the male reproductive organ in a flower. The specification of stamen identity in Arabidopsis (Arabidopsis thaliana) is controlled by a combination of the B genes APETALA3 (AP3) and PISTILLATA, the C gene AGAMOUS (AG), and the E genes SEPALLATA1 (SEP1) to SEP4. The "floral organ-building" gene SPOROCYTELESS/NOZZLE (SPL/NZZ) plays a central role in regulating anther cell differentiation. However, much less is known about how "floral organ identity" and floral organ-building genes interact to control floral organ development. In this study, we report that ectopic expression of SPL/NZZ not only affects flower development in the wild-type background but also leads to the transformation of petal-like organs into stamen-like organs in flowers of ap2-1, a weak ap2 mutant allele. Moreover, our loss-of-function analysis indicates that the spl/nzz mutant enhances the phenotype of the ag weak allele ag-4. Furthermore, ectopic expression and overexpression of SPL/NZZ altered expression of AG, SEP3, and AP2 in rosette leaves and flowers, while ectopic expression of SPL/NZZ resulted in ectopic expression of AG and SEP3 in the outer whorls of flowers. Our results indicate that the SPL/NZZ gene is engaged in controlling stamen identity via interacting with genes required for stamen identity in Arabidopsis.

A Leaky Mutation in DWARF4 Reveals an Antagonistic Role of Brassinosteroid in the Inhibition of Root Growth by Jasmonate in Arabidopsis

Tue, 03 Nov 2009 12:22:27 PST

The F-box protein CORONATINE INSENSITIVE1 (COI1) plays a central role in jasmonate (JA) signaling and is required for all JA responses in Arabidopsis (Arabidopsis thaliana). To dissect JA signal transduction, we isolated the partially suppressing coi1 (psc1) mutant, which partially suppressed coi1 insensitivity to JA inhibition of root growth. The psc1 mutant partially restored JA sensitivity in coi1-2 background and displayed JA hypersensitivity in wild-type COI1 background. Genetic mapping, sequence analysis, and complementation tests revealed that psc1 is a leaky mutation of DWARF4 (DWF4) that encodes a key enzyme in brassinosteroid (BR) biosynthesis. Physiological analysis showed that an application of exogenous BR eliminated the partial restoration of JA sensitivity by psc1 in coi1-2 background and the JA hypersensitivity of psc1 in wild-type COI1 background. Exogenous BR also attenuated JA inhibition of root growth in the wild type. In addition, the expression of DWF4 was inhibited by JA, and this inhibition was dependent on COI1. These results indicate that (1) BR is involved in JA signaling and negatively regulates JA inhibition of root growth, and (2) the DWF4 is down-regulated by JA and is located downstream of COI1 in the JA-signaling pathway.

Functional Analysis of {alpha}-DOX2, an Active {alpha}-Dioxygenase Critical for Normal Development in Tomato Plants

Tue, 03 Nov 2009 12:22:27 PST

Plant -dioxygenases initiate the synthesis of oxylipins by catalyzing the incorporation of molecular oxygen at the -methylene carbon atom of fatty acids. Previously, -DOX1 has been shown to display -dioxygenase activity and to be implicated in plant defense. In this study, we investigated the function of a second -dioxygenase isoform, -DOX2, in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). Recombinant Sl-DOX2 and At-DOX2 proteins catalyzed the conversion of a wide range of fatty acids into 2(R)-hydroperoxy derivatives. Expression of Sl-DOX2 and At-DOX2 was found in seedlings and increased during senescence induced by detachment of leaves. In contrast, microbial infection, earlier known to increase the expression of -DOX1, did not alter the expression of Sl-DOX2 or At-DOX2. The tomato mutant divaricata, characterized by early dwarfing and anthocyanin accumulation, carries a mutation at the Sl-DOX2 locus and was chosen for functional studies of -DOX2. Transcriptional changes in such mutants showed the up-regulation of genes playing roles in lipid and phenylpropanoid metabolism, the latter being in consonance with the anthocyanin accumulation. Transgenic expression of At-DOX2 and Sl-DOX2 in divaricata partially complemented the compromised phenotype in mature plants and fully complemented it in seedlings, thus indicating the functional exchangeability between -DOX2 from tomato and Arabidopsis. However, deletion of At-DOX2 in Arabidopsis plants did not provoke any visible phenotypic alteration indicating that the relative importance of -DOX2 in plant physiology is species specific.

CHOTTO1, a Putative Double APETALA2 Repeat Transcription Factor, Is Involved in Abscisic Acid-Mediated Repression of Gibberellin Biosynthesis during Seed Germination in Arabidopsis

Yano, R., Kanno, Y., Jikumaru, Y., Nakabayashi, K., Kamiya, Y., Nambara, E. — Thu, 01 Oct 2009 06:20:40 PDT

The phytohormones abscisic acid (ABA) and gibberellins (GAs) are the primary signals that regulate seed dormancy and germination. In this study, we investigated the role of a double APETALA2 repeat transcription factor, CHOTTO1 (CHO1), in seed dormancy, germination, and phytohormone metabolism of Arabidopsis (Arabidopsis thaliana). Wild-type seeds were dormant when freshly harvested seeds were sown, and these seeds were released from dormancy after a particular period of dry storage (after-ripening). The cho1 mutant seeds germinated easily even in a shorter period of storage than wild-type seeds. The cho1 mutants showed reduced responsiveness to ABA, whereas transgenic plants constitutively expressing CHO1 (p35S::CHO1) showed an opposite phenotype. Notably, after-ripening reduced the ABA responsiveness of the wild type, cho1 mutants, and p35S::CHO1 lines. Hormone profiling demonstrated that after-ripening treatment decreased the levels of ABA and salicylic acid and increased GA₄, jasmonic acid, and isopentenyl adenine when wild-type seeds were imbibed. Expression analysis showed that the transcript levels of genes for ABA and GA metabolism were altered in the wild type by after-ripening. Hormone profiling and expression analyses indicate that cho1 seeds, with a short period of storage, resembled fully after-ripened wild-type seeds. Genetic analysis showed that the cho1 mutation partially restored delayed seed germination and reduced GA biosynthesis activity in the ABA-overaccumulating cyp707a2-1 mutant background but did not restore seed germination in the GA-deficient ga1-3 mutant background. These results indicate that CHO1 acts downstream of ABA to repress GA biosynthesis during seed germination.

The Arabidopsis GRF-INTERACTING FACTOR Gene Family Performs an Overlapping Function in Determining Organ Size as Well as Multiple Developmental Properties

Lee, B. H., Ko, J.-H., Lee, S., Lee, Y., Pak, J.-H., Kim, J. H. — Thu, 01 Oct 2009 06:20:40 PDT

Previously, the GRF-INTERACTING FACTOR1 (GIF1)/ANGUSTIFOLIA3 (AN3) transcription coactivator gene, a member of a small gene family comprising three genes, was characterized as a positive regulator of cell proliferation in lateral organs, such as leaves and flowers, of Arabidopsis (Arabidopsis thaliana). As yet, it remains unclear how GIF1/AN3 affects the cell proliferation process. In this study, we demonstrate that the other members of the GIF gene family, GIF2 and GIF3, are also required for cell proliferation and lateral organ growth, as gif1, gif2, and gif3 mutations cause a synergistic reduction in cell numbers, leading to small lateral organs. Furthermore, GIF1, GIF2, and GIF3 overexpression complemented a cell proliferation defect of the gif1 mutant and significantly increased lateral organ growth of wild-type plants as well, indicating that members of the GIF gene family are functionally redundant. Kinematic analysis on leaf growth revealed that the gif triple mutant as well as other strong gif mutants developed leaf primordia with fewer cells, which was due to the low rate of cell proliferation, eventually resulting in earlier exit from the proliferative phase of organ growth. The low proliferative activity of primordial leaves was accompanied by decreased expression of cell cycle-regulating genes, indicating that GIF genes may act upstream of cell cycle regulators. Analysis of gif double and triple mutants clarified a previously undescribed role of the GIF gene family: gif mutants had small vegetative shoot apical meristems, which was correlated with the development of small leaf primordia. gif triple mutants also displayed defective structures of floral organs. Taken together, our results suggest that the GIF gene family plays important roles in the control of cell proliferation via cell cycle regulation and in other developmental properties that are associated with shoot apical meristem function.

BRASSINOSTEROID UPREGULATED1, Encoding a Helix-Loop-Helix Protein, Is a Novel Gene Involved in Brassinosteroid Signaling and Controls Bending of the Lamina Joint in Rice

Thu, 01 Oct 2009 06:20:40 PDT

Brassinosteroids (BRs) are involved in many developmental processes and regulate many subsets of downstream genes throughout the plant kingdom. However, little is known about the BR signal transduction and response network in monocots. To identify novel BR-related genes in rice (Oryza sativa), we monitored the transcriptomic response of the brassinosteroid deficient1 (brd1) mutant, with a defective BR biosynthetic gene, to brassinolide treatment. Here, we describe a novel BR-induced rice gene BRASSINOSTEROID UPREGULATED1 (BU1), encoding a helix-loop-helix protein. Rice plants overexpressing BU1 (BU1:OX) showed enhanced bending of the lamina joint, increased grain size, and resistance to brassinazole, an inhibitor of BR biosynthesis. In contrast to BU1:OX, RNAi plants designed to repress both BU1 and its homologs displayed erect leaves. In addition, compared to the wild type, the induction of BU1 by exogenous brassinolide did not require de novo protein synthesis and it was weaker in a BR receptor mutant OsbriI (Oryza sativa brassinosteroid insensitive1, d61) and a rice G protein alpha subunit (RGA1) mutant d1. These results indicate that BU1 protein is a positive regulator of BR response: it controls bending of the lamina joint in rice and it is a novel primary response gene that participates in two BR signaling pathways through OsBRI1 and RGA1. Furthermore, expression analyses showed that BU1 is expressed in several organs including lamina joint, phloem, and epithelial cells in embryos. These results indicate that BU1 may participate in some other unknown processes modulated by BR in rice.

Analysis of PHOTOPERIOD SENSITIVITY5 Sheds Light on the Role of Phytochromes in Photoperiodic Flowering in Rice

Andres, F., Galbraith, D. W., Talon, M., Domingo, C. — Thu, 01 Oct 2009 06:20:40 PDT

A great number of plants synchronize flowering with day length. In rice (Oryza sativa), photoperiod is the primary environmental cue that triggers flowering. Here, we show that the s73 mutant, identified in a -irradiated Bahia collection, displays early flowering and photoperiodic insensitivity due to a null mutation in the PHOTOPERIOD SENSITIVITY5 (SE5) gene, which encodes an enzyme implicated in phytochrome chromophore biosynthesis. s73 mutant plants show a number of alterations in the characteristic diurnal expression patterns of master genes involved in photoperiodic control of flowering, resulting in up-regulation of the floral integrator Heading date3a (Hd3a). Early heading date1 (Ehd1), an additional rice floral activator, was also highly expressed in the s73 mutant, suggesting that SE5 represses Ehd1 in wild-type plants. Silencing of Ehd1 in both Bahia and s73 backgrounds indicated that SE5 regulates Ehd1 expression. The data also indicate that SE5 confers photoperiodic sensitivity through regulation of Hd1. These results provide direct evidence that phytochromes inhibit flowering by affecting both Hd1 and Ehd1 flowering pathways.

SAUR39, a Small Auxin-Up RNA Gene, Acts as a Negative Regulator of Auxin Synthesis and Transport in Rice

Kant, S., Bi, Y.-M., Zhu, T., Rothstein, S. J. — Thu, 01 Oct 2009 06:20:40 PDT

The phytohormone auxin plays a critical role for plant growth by regulating the expression of a set of genes. One large auxin-responsive gene family of this type is the small auxin-up RNA (SAUR) genes, although their function is largely unknown. The expression of the rice (Oryza sativa) SAUR39 gene showed rapid induction by transient change in different environmental factors, including auxin, nitrogen, salinity, cytokinin, and anoxia. Transgenic rice plants overexpressing the SAUR39 gene resulted in lower shoot and root growth, altered shoot morphology, smaller vascular tissue, and lower yield compared with wild-type plants. The SAUR39 gene was expressed at higher levels in older leaves, unlike auxin biosynthesis, which occurs largely in the meristematic region. The transgenic plants had a lower auxin level and a reduced polar auxin transport as well as the down-regulation of some putative auxin biosynthesis and transporter genes. Biochemical analysis also revealed that transgenic plants had lower chlorophyll content, higher levels of anthocyanin, abscisic acid, sugar, and starch, and faster leaf senescence compared with wild-type plants at the vegetative stage. Most of these phenomena have been shown to be negatively correlated with auxin level and transport. Transcript profiling revealed that metabolic perturbations in overexpresser plants were largely due to transcriptional changes of genes involved in photosynthesis, senescence, chlorophyll production, anthocyanin accumulation, sugar synthesis, and transport. The lower growth and yield of overexpresser plants was largely recovered by exogenous auxin application. Taken together, the results suggest that SAUR39 acts as a negative regulator for auxin synthesis and transport.

The TRANSPORT INHIBITOR RESPONSE2 Gene Is Required for Auxin Synthesis and Diverse Aspects of Plant Development

Yamada, M., Greenham, K., Prigge, M. J., Jensen, P. J., Estelle, M. — Wed, 02 Sep 2009 10:00:32 PDT

The plant hormone auxin plays an essential role in plant development. However, only a few auxin biosynthetic genes have been isolated and characterized. Here, we show that the TRANSPORT INHIBITOR RESPONSE2 (TIR2) gene is required for many growth processes. Our studies indicate that the tir2 mutant is hypersensitive to 5-methyl-tryptophan, an inhibitor of tryptophan synthesis. Further, treatment with the proposed auxin biosynthetic intermediate indole-3-pyruvic acid (IPA) and indole-3-acetic acid rescues the tir2 short hypocotyl phenotype, suggesting that tir2 may be affected in the IPA auxin biosynthetic pathway. Molecular characterization revealed that TIR2 is identical to the TAA1 gene encoding a tryptophan aminotransferase. We show that TIR2 is regulated by temperature and is required for temperature-dependent hypocotyl elongation. Further, we find that expression of TIR2 is induced on the lower side of a gravitropically responding root. We propose that TIR2 contributes to a positive regulatory loop required for root gravitropism.

The Paralogous Genes RADICAL-INDUCED CELL DEATH1 and SIMILAR TO RCD ONE1 Have Partially Redundant Functions during Arabidopsis Development

Teotia, S., Lamb, R. S. — Wed, 02 Sep 2009 10:00:32 PDT

RADICAL-INDUCED CELL DEATH1 (RCD1) and SIMILAR TO RCD ONE1 (SRO1) are the only two proteins encoded in the Arabidopsis (Arabidopsis thaliana) genome containing both a putative poly(ADP-ribose) polymerase catalytic domain and a WWE protein-protein interaction domain, although similar proteins have been found in other eukaryotes. Poly(ADP-ribose) polymerases mediate the attachment of ADP-ribose units from donor NAD⁺ molecules to target proteins and have been implicated in a number of processes, including DNA repair, apoptosis, transcription, and chromatin remodeling. We have isolated mutants in both RCD1 and SRO1, rcd1-3 and sro1-1, respectively. rcd1-3 plants display phenotypic defects as reported for previously isolated alleles, most notably reduced stature. In addition, rcd1-3 mutants display a number of additional developmental defects in root architecture and maintenance of reproductive development. While single mutant sro1-1 plants are relatively normal, loss of a single dose of SRO1 in the rcd1-3 background increases the severity of several developmental defects, implying that these genes do share some functions. However, rcd1-3 and sro1-1 mutants behave differently in several developmental events and abiotic stress responses, suggesting that they also have distinct functions. Remarkably, rcd1-3; sro1-1 double mutants display severe defects in embryogenesis and postembryonic development. This study shows that RCD1 and SRO1 are at least partially redundant and that they are essential genes for plant development.

Molecular and Biochemical Characterization of AtPAP15, a Purple Acid Phosphatase with Phytase Activity, in Arabidopsis

Kuang, R., Chan, K.-H., Yeung, E., Lim, B. L. — Wed, 02 Sep 2009 10:00:32 PDT

Purple acid phosphatase (PAP) catalyzes the hydrolysis of phosphate monoesters and anhydrides to release phosphate within an acidic pH range. Among the 29 PAP-like proteins in Arabidopsis (Arabidopsis thaliana), AtPAP15 (At3g07130) displays a greater degree of amino acid identity with soybean (Glycine max; GmPHY) and tobacco (Nicotiana tabacum) PAP (NtPAP) with phytase activity than the other AtPAPs. In this study, transgenic Arabidopsis that expressed an AtPAP15 promoter::β-glucuronidase (GUS) fusion protein showed that AtPAP15 expression was developmentally and temporally regulated, with strong GUS staining at the early stages of seedling growth and pollen germination. The expression was also organ/tissue specific, with strongest GUS staining in the vasculature, pollen grains, and roots. The recombinant AtPAP purified from transgenic tobacco exhibited broad substrate specificity with moderate phytase activity. AtPAP15 T-DNA insertion lines exhibited a lower phytase and phosphatase activity in seedling and germinating pollen and lower pollen germination rate compared with the wild type and their complementation lines. Therefore, AtPAP15 likely mobilizes phosphorus reserves in plants, particularly during seed and pollen germination. Since AtPAP15 is not expressed in the root hair or in the epidermal cells, it is unlikely to play any role in external phosphorus assimilation.

Null Mutation of the MdACS3 Gene, Coding for a Ripening-Specific 1-Aminocyclopropane-1-Carboxylate Synthase, Leads to Long Shelf Life in Apple Fruit

Wed, 02 Sep 2009 10:00:33 PDT

Expression of MdACS1, coding for 1-aminocyclopropane-1-carboxylate synthase (ACS), parallels the level of ethylene production in ripening apple (Malus domestica) fruit. Here we show that expression of another ripening-specific ACS gene (MdACS3) precedes the initiation of MdACS1 expression by approximately 3 weeks; MdACS3 expression then gradually decreases as MdACS1 expression increases. Because MdACS3 expression continues in ripening fruit treated with 1-methylcyclopropene, its transcription appears to be regulated by a negative feedback mechanism. Three genes in the MdACS3 family (a, b, and c) were isolated from a genomic library, but two of them (MdACS3b and MdACS3c) possess a 333-bp transposon-like insertion in their 5' flanking region that may prevent transcription of these genes during ripening. A single nucleotide polymorphism in the coding region of MdACS3a results in an amino acid substitution (glycine-289 -> valine) in the active site that inactivates the enzyme. Furthermore, another null allele of MdACS3a, Mdacs3a, showing no ability to be transcribed, was found by DNA sequencing. Apple cultivars homozygous or heterozygous for both null allelotypes showed no or very low expression of ripening-related genes and maintained fruit firmness. These results suggest that MdACS3a plays a crucial role in regulation of fruit ripening in apple, and is a possible determinant of ethylene production and shelf life in apple fruit.

Interactions between Auxin and Strigolactone in Shoot Branching Control

Hayward, A., Stirnberg, P., Beveridge, C., Leyser, O. — Wed, 02 Sep 2009 10:00:33 PDT

In Arabidopsis (Arabidopsis thaliana), the carotenoid cleavage dioxygenases MORE AXILLARY GROWTH3 (MAX3) and MAX4 act together with MAX1 to produce a strigolactone signaling molecule required for the inhibition of axillary bud outgrowth. We show that both MAX3 and MAX4 transcripts are positively auxin regulated in a manner similar to the orthologous genes from pea (Pisum sativum) and rice (Oryza sativa), supporting evolutionary conservation of this regulation in plants. This regulation is important for branching control because large auxin-related reductions in these transcripts are associated with increased axillary branching. Both transcripts are up-regulated in max mutants, and consistent with max mutants having increased auxin in the polar auxin transport stream, this feedback regulation involves auxin signaling. We suggest that both auxin and strigolactone have the capacity to modulate each other's levels and distribution in a dynamic feedback loop required for the coordinated control of axillary branching.