Chen, Y.
Citation
Chen, Y. (2011, December 20). Functional analysis of genes involved in the regulation of development of reproductive organs in rice (Oryza sativa). Retrieved from
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Functions of OsJAR1 in Floret Opening and Anther Dehiscence in Rice (Oryza sativa)
Yuguo Xiao*1, 2, Yi Chen*1,Jeroen Heijmans1, Angela Hoogenboom1, Tatsiana Charnikhova3, Patrick P.J. Mulder4, Adamantia Agalou1,Zhen Zhu2, Mei Wang1,5, Ludovico Dreni6, Martin M. Kater6, Corinne Michel7, Jean-Benoit
Morel7, Harro Bouwmeester3, and Pieter B.F. Ouwerkerk1
1 Department of Molecular and Developmental Genetics, Institute Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, PO Box 9505, 2300 RA Leiden, The Netherlands
2 State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No.1 West Beichen Road, Chaoyang District, Beijing, 100101 China
3 Plant Research International BV, Droevendaalsesteeg 1 6708 PB, PO Box 16, 6700 AA, Wageningen, The Netherlands
4 RIKILT– Institute of Food Safety, Bornsesteeg 45, 6708 PD, PO Box 230 6700 AE, Wageningen, The Netherlands
5 SU BioMedicine-TNO, Utrechtseweg 48, 3704 HE Zeist, PO Box 360, 3700 AJ Zeist, The Netherlands
6 Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita` degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
7 UMR BGPI INRA/CIRAD/SupAgro, Campus International de Baillarguet, TA A 54 / K34398 Montpellier, France
* These authors contribute equally to the work
79 Abstract
In literature two rice JASMONATE RESISTANT 1 (osjar1) lines were described that were mutated by the endogenous retrotransposon Tos17. The gene disrupted in the two independent lines, named osjar1-2 and osjar1-3 respectively, is predicted to encode a putative auxin-responsive GRETCHEN HAGEN 3 (GH3) family protein named OsJAR1. In this chapter, we present a detailed characterization of the osjar1 phenotype and present a model for the biological functions of OsJAR1.
In the osjar1 mutants, the florets fail to close again after opening because the withering of the lodicules is delayed by several days whereas this normally happens within hours. The reduced fertility of osjar1 plants is caused by a delay in anther dehiscence. Normally, OsJAR1 mRNA is present in floral and vegetative organs but is down-regulated in osjar1-2 and the length is shorter in osjar1-3 due to the Tos17 insertions. In contrast to the wild type, floret opening in osjar1-2 and osjar1-3 cannot be induced by exogenous application of methyl jasmonate (MeJA) and root growth is not inhibited by MeJA. Overexpression of OsJAR1 under control of the rice actin promoter rescued the osjar1-2 phenotype and restored floret opening in response to MeJA. osjar1-3 is more sensitive to rice blast disease and overexpression of OsJAR1 in wild type rice plants increases resistance to blast disease, indicating a role of OsJAR1 in defence against pathogens. The OsJAR1 protein specifically conjugates alanine, cysteine, leucine, isoleucine, methionine, phenylalanine, tryptophan and valine to JA in in vitro assays and JA-Ile levels were reduced in osjar1-2 plants. We conclude that OsJAR1 is a JA-amino acid synthetase that is required to convert JA into JA-Ile for optimal flower opening, anther dehiscence, seed development and resistance to blast disease.
Introduction
Anthesis is the period during which a flower is fully open and functional and is obviously an essential step in the production of progeny. Events that take place during anthesis include flower opening and closure, anther dehiscence, release of pollen grains and fertilization. In general, flower opening and closure is caused by the movement of flower organs. In tulip and taraxacum, for example, petal movement due to unequal growth on both sides of the petals results in the opening of flower (Tanaka et al., 1988; Van Doorn and Van Meeteren, 2003). In rice, the opening and closure of the florets result from the separation of lemma from palea caused by the expansion and subsequent atrophy or withering of the lodicules (Matsuo and Hoshikawa, 1993; Qin et al., 2005). External factors such as light (Kaihara and Takimoto, 1980, 1981; Bieleski et al., 2000) and temperature (Kaihara and Takimoto, 1981; Ichimura and Suto, 1998) were described to have effects on flower opening and closure in several plant species. The plant hormones ethylene and MeJA were also shown to have critical functions in regulation of flower opening and closure. Ethylene promotes or inhibits flower opening depending on the species. For instance, in cut roses, flower opening is promoted by exogenous ethylene application (Ma et al., 2005). MeJA induces floret opening in rice and the numbers of induced opening florets are correlated with MeJA concentration in the wild type (Zeng et al., 1999). Thus far, no gene directly related to regulation of flower opening and closure has been identified.
Anther dehiscence and pollen grain release is another important step during anthesis. Several genes involved in anther dehiscence have been identified and characterized in several plant species.
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These genes include, AOS/DDE2-2 (Park et al., 2002; Von Malek et al., 2002), AtMYB26 (Steiner- Lange et al., 2003), COI1 (Xie et al., 1998), DAD1 (Ishiguro et al., 2001), NST1/NST2 (Mitsuda et al., 2005), OPR3/DDE1 (Sanders et al., 2000; Stintzi and Browse, 2000) and RPK2 (Mizuno et al., 2007) from Arabidopsis and NEC1 (Ge et al., 2001) and PhETR2 (Wang and Kumar, 2007) from petunia. Loss-of-function mutants of these genes show obvious anther developmental defects in timing of anther dehiscence, filament elongation or pollen viability. AOS/DDE2-2, COI1, DAD1 and OPR3/DDE1 encode proteins involved in the jasmonic acid (JA) pathway, which indicates that this plant hormone may be a critical regulator of anther dehiscence in plants. In rice, the Myb domain protein AID1 (Zhu et al., 2004) was shown to be involved in anther development. The aid1 mutation gives rise to partial completion of anther indehiscence and this phenotype could not be complemented by exogenous application of MeJA, suggesting that AID1 is not involved in MeJA- mediated anther dehiscence in rice.
Riemann et al. (2008) described two Tos17 mutants named osjar1 and osjar2 respectively after JASMONATE RESISTANT 1. The gene disrupted in two independent lines osjar1-2 and osjar1- 3 is predicted to encode a putative auxin-responsive GRETCHEN HAGEN 3 (GH3) family protein which is closely related to JAR1 of Nicotiana glutinosa (NgJAR1) and JAR1 of Arabidopsis (AtJAR1). However the relation between the osjar1 phenotype and the biological function of OsJAR1 remained unclear. Simultaneously we identified the same osjar1 mutant in a population of T-DNA and transposon mutants in genes predominantly expressed in developing flowers and grains.
The osjar1 lines showed a very obvious phenotype of open-staying flowers and elongated seeds. In this chapter we report on a detailed characterization of the osjar1 phenotype and propose a biological function of OsJAR1. In wild type plants, OsJAR1 mRNA is present in both floral and vegetative organs, but it is down-regulated in osjar1-2 whereas its length is shorter in osjar1-3.
Analysis of an OsJAR1 promoter-β-glucuronidase construct revealed that the OsJAR1 promoter is predominantly active in the vasculature of floral and vegetative tissues. We confirmed localization of OsJAR1 expression in anthers using RNA in situ hybridization. Floret opening in osjar1-2 and osjar1-3 cannot be induced by exogenous application of MeJA which demonstrated that MeJA- induced floret opening pathway is defective in osjar1-2 and osjar1-3. Overexpression of OsJAR1 in the background of osjar1-2 rescued the osjar1-2 phenotype. osjar1-2 mutants show reduced root growth inhibition by MeJA which suggested that OsJAR1 is probably also involved in other aspects of MeJA-mediated plant growth and developmental processes. In addition, we observed that osjar1- 3 has reduced resistance to the blast fungus M. oryzae and overexpression of OsJAR1 in control rice plants enhanced resistance towards blast disease, confirming a link between JA and disease resistance. Thin-layer chromatography (TLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of products produced in vitro by recombinant OsJAR1 protein demonstrated that this GH3 protein is able to form conjugates between JA and alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, tryptophan and valine but not with the twelve other amino acids.
In osjar1-2 we observed a significantly decreased level of JA-Ile in both JA treated and untreated leaves which confirm the function of OsJAR1 in conjugation of JA to isoleucine. We propose that this step and JA-Ile plays a crucial role in timing of flower opening and closure, anther dehiscence and disease resistance.
81 Results
Phenotypical characterization of osjar1
Normally in rice, anthesis only happens during a specific period of the day and lasts for 40 to 90 min depending on the cultivar. In our greenhouse settings, florets of rice Nipponbare rice plants open at noon and keep opened for approximately 90 minutes. During anthesis, florets open and the filaments of the stamen elongate, so that the anthers are positioned outside the florets; simultaneously anther dehiscence and pollination are taking place. At the end of anthesis, florets are closing again (Figure 1A-1E). The opening and closure of the florets is driven by the expansion and subsequently atrophy of the lodicules. The rice floret has two lodicules just below the base of its stamina and on the lemma side. The lodicules swell just before the anthesis, pushing the lemma away resulting in the opening of the florets. At the end of anthesis, closure of rice florets is due to a quick shrinking of lodicules due to withering (Figure 1K-1M) (Matsuo and Hoshikawa, 1993). In a population of Tos17
Figure 1. Comparison of the phenology of florets from wild-type and osjar1-2.plants. A-E. Time course of floret anthesis in wild type plants. Photos were taken just before anthesis or 15, 30, 60 and 90 minutes after the floret started to open, respectively. F-J. Time course of floret anthesis in osjar1-2. Photos were taken just before anthesis or 2 hours, one day, three days and five days after the floret started to open, respectively. K-M.
Close-up of the lodicules of the wild-type were monitored just before floret opening (K), during floret opening (L) and just the end of florets opening (M). P-R. Close-up of lodicules of osjar1-2 were monitored just before floret opening (P), during floret opening (Q) and three days after florets started to open (R), respectively. N.
Comparison of spikelet in the wild type and osjar1-2. From left to right, mature osjar1-2 spikelet, osjar1-2 spikelet at the filling stage and mature wild type spikelet. O. Comparison of wild type grains and osjar1-2 grains. From left to right, osjar1-2 grains at the filling stage, mature osjar1-2 grains, wild type grains at the filling stage and mature wild type grains. S. Wild type mature grains. T. osjar1-2 mature grains. lo, lodicules.
Bars (A-J, N, O, S, T) = 1 mm; Bars (K-M, P-R) =0.5 mm.
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Figure 2. Comparison of anthers from wild type and osjar1-2 plants. A and B. Floret organs of wild type (A) and osjar1 (B) just before floret opening. C. Stamen of the wild-type (a) and the osjar1-2 mutant (b and c) just before floret opening. D. Stamen of the wild-type (d and e) and the osjar1-2 mutant (f and g) during floret opening. E. Wild-type anther at the pollen mature stage before anther dehiscence. F. Dehisced wild type anther.
G. osjar1-2 anther at the pollen mature stage before floret opening. H and I. osjar1-2 anther during floret opening. J-N. Close-up of the region including the connective of anther in E-I, respectively. en, endothelium;
ca, cavity; ccc, circular cell cluster; se, septum; ep, epidermis; P, pollen; V, vascular bundle. Bars (A-B) = 1 mm; Bars (C-D) = 100 μm; Bars (E-I) = 50 μm; Bars (J-N) = 25 μm.
mutants, two different osjar1 mutants were isolated which were characterized by an obvious open staying phenotype of the flower (Riemann et al., 2008). In a detailed study we observed ourselves in total five major differences between control plants and osjar1. First, the opening of the osjar1 florets happens at random, which means also during the night instead of in a certain period of the day as normally occurs with control flowers. Second, osjar1 florets failed to close after the opening (Figure 1G-1J). Third, 87.2% of the anthers in osjar1 are not dehiscing (Figure 1G-1J, 2D). Fourth, senescence of the floral organs of osjar1 is delayed compared to control plants. For instance, in osjar1 florets, the lodicules are still big enough to keep the floret open even three days after the floret starts open (Figure 1R) whereas wild type florets already close after 90 minutes (Figure 1M).
Fifth, osjar1 plants are less fertile and have abnormal seed morphology (Figure 1N, O, S and T).
Only 7.5-14.5% of osjar1 florets have seeds set and the vast majority of florets are sterile. The osjar1 mutation is in cultivar Nipponbare, which is a Japonica type and has thick and round seeds (Figure 1O and 1S). In contrast, osjar1 grains are narrower and longer (Figure 1O and 1T). More than 99% of the grains from osjar1 homozygous plants show this phenotype. In addition, grains from osjar1 plants show wrinkled, curved and over-dried endosperm and the withered stigma is still kept attached to the mature grain (Figure 1N and 1O). Compared to the wild type, the osjar1 anther is shorter and a little crooked (Figure 2 A-C). Normally, the anther wall consists of four cell layers
83 from outside to inside, epidermis and endothecium, middle layer and tapetum (Figure 2E, 2J).
Adjacent to the circular cell cluster there is a dehiscent cavity present, which is a single layer of specialized epidermal cells connecting each other of the pair of loculi and is the final breakage site of anther dehiscence (Figure 2J) (Zhu et al., 2004). The endothelium close to the cavity and circular cell cluster shows obvious well-developed U-shaped cell wall thickenings, which is considered to be important for anther dehiscence (Figure 2J) (Matsui et al., 1999). During floret opening, the pollen increase to swell resulting in increased pressure and simultaneously cells in the anther wall lose water. As a result, the anther wall gets twisted and the circular cell cluster splits resulting in the rupture of anther loculi and pollen release (Figure 2D, 2F and 2K) (Matsui et al., 1999). Cross sections of osjar1-2 anther show no obvious difference except that the cell shape of several cells during the stage just before floret opening and anther dehiscence is different. That is, the epidermis cells, endothelium cells and septum cells in osjar1 anther are more irregular than normally (Figure 2E, 2G, 2J and 2L). In osjar1, 87.2% of the osjar1-2 anthers are not or only partly dehisced after floret opening (Figure 2D, 2H and 2I). In the non-dehisced anthers, the circular cell cluster was still kept intact and not splitted although the anther wall was shrunken considerably (Figure 2H, 2M).
The osjar1 phenotype co-segregates with a Tos17 insertion in the OsJAR1 gene and correlates with the occurrence of abnormal transcripts in mutants osjar1-2 and osjar1-3
The osjar1 phenotype was found in populations derived from Tos17 insertion lines NC2728 (osjar1- 2) and ND0051 (osjar1-3), which originated from the Hirochika Laboratory (Miyao et al., 2003).
Three out of 19 NC2728 plants and four out of 16 ND0051 plants displayed the osjar1 phenotype.
All progeny of three osjar1-2 plants (NC2728-7, 14, 19) exhibited an identical phenotype. The phenotypic segregation in line NC2728 was according to a ratio of three wild type versus one mutant plants. The same result was obtained in line osjar1-3 which demonstrates that the osjar1 phenotype is very likely caused by a recessive mutation. Database searches at NCBI (http://www.ncbi.nlm.nih.gov) retrieved 16 FSTs (flanking sequence tags) from mutant lines NC2728 and ND0051. By BLASTing these with the rice genomic database in TIGR (http://rice.plantbiology.msu.edu/), only FSTs T06540T and ND0051_0_403_1A from lines NC2728 and ND0051 respectively, were found to be associated with locus LOC_Os05g50890. This locus is predicted to contain five exons and encodes a GH3 family protein (Jain et al., 2006) named OsJAR1 (Riemann et al., 2008). The Tos17 alleles in mutant lines NC2728 and ND0051 were found to be inserted into exon 3 and 4 of OsJAR1, respectively (Figure 3A). Because these two mutants show the same phenotype, we hypothesized that it is caused by Tos17 insertions in this locus leading to disruption of gene function. This hypothesis was strongly supported by Southern blot hybridization of XbaI-digested genomic DNA from wild type plants and plants from the two mutant populations (lines NC2728 and ND0051) with OsJAR1 specific probes. The wild type plants were predicted to contain a hybridizing restriction fragment of 2.5 kb and 1.2 kb. Because there is an XbaI site in the Tos17 retrotransposon, homozygous Tos17 insertion plants were predicted to contain a fragment of 5 kb in mutant line NC2728 and a 2.2 kb fragment in ND0051 including a partial Tos17 fragment which is in agreement with the Southern blot hybridization results (Figure 3D to F). Because only homozygous Tos17 insertion plants (Tos17/Tos17) in lines NC2728 and ND0051 show the osjar1 phenotype, this result confirms that the osjar1 phenotype is co-segregating with the Tos17 insertion in OsJAR1. This was confirmed by comparing the phenotype and genotype of the first
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Figure 3. Association of the osjar1 phenotype with Tos17 insertions in OsJAR1. A. Schematic structure of OsJAR1 and positions of Tos17. OsJAR1 contains five exons (black bars) and four introns (white bars). ATG and TGA indicate start and stop codons respectively. Triangles indicate the positions of Tos17 insertions in mutant line NC2728, NE8577 and ND0051 respectively. Black arrows indicate the orientations of the Tos17 insertions. The lines below the OsJAR1 gene indicate the positions of the probes used in Southern and northern blot analyses (P1, P2 and N1, N2, respectively). Xb, XbaI. B and C. Positions and orientations of Tos17 in osjar1-2 (B) and osjar1-3 (C). Letters in grey indicatedthe repeated sequence caused by Tos17 insertion, black letters indicate nucleotides from exon 4 and exon 5 of ogm1 and ogm2, respectively. D, E and F. Co- segregation of Tos17 in OsJAR1 with the mutant phenotype of osjar1. As a result of Tos17 insertions, 5 kb, 2.2 kb and 2.5 kb polymorphic bands appear in mutant lines NC2728, ND0051 and NE8544, respectively, whereas the wild-type OsJAR1 gene showed a 2.5 kb, 1.2 kb and 1.2 kb band in segregatns of mutant line NC2728, ND0051 and NE8544, respectively. The 5 kb and 2.2 kb polymorphic bands in mutant lines NC2728 and ND0051 were fully associated with the osjar1 phenotype (O). W, OsJAR1 in a wild type plant. H, heterozygous OsJAR1/osjar1 plant. P, wild-type Nipponbare control. G. OsJAR1 mRNA expression in wild type and osjar1 mutants. Northern blot hybridized with probe N2 and N1 which were indicated in Figure 3A.
Blots were hybridized with 17S rRNA to check for the total RNA loading. L leaves; S stems; R, roots; P, panicles.
generation of heterozygous Tos17 insertion plants in both mutant lines. The progeny of heterozygous Tos17 insertion plants segregated at a 1:2:1 ratio of homozygous (Tos17/Tos17), heterozygous (Tos17/+) and Tos17 null (+/+) and only Tos17/Tos17 plants display the osjar1 phenotype. The Tos17 insertion positions in osjar1-2 and osjar1-3 were confirmed by sequencing the FSTs of the Tos17 insertions in osjar1-2 and osjar1-3 genomic DNA with two primers, one is OsJAR1-specific and adjacent to the annotated Tos17 insertion site, and the other is Tos17 specific. The sequencing results on the FSTs show that the Tos17 retrotransposons in the osjar1-2 and osjar1-3 mutants were inserted after amino acid 354 and 498 of the OsJAR1 ORF, respectively. FST sequences obtained from osjar1-2 and osjar1-3 were submitted to NCBI with Genbank Accession codes FI494725-FI494726 and FI494727-FI494728, respectively. The insertions in osjar1-2 and osjar1-3 caused a duplication
85 of five nucleotides OsJAR1 target sequence adjacent to the border of Tos17 (Figure 3B-3C).
To check the expression of the OsJAR1 gene in osjar1-2, osjar1-3 and control plants, northern blot analysis was performed using two OsJAR1-specific probes named N1 and N2 (Figure 3A). With probe N1, a 2.4 kb transcript was detected in different tissues including root, shoot, leaf and young panicle in the wild type, but similar sized transcripts were not detected in osjar1-2 and osjar1-3 (Figure 3G). For osjar1-2, we only observed a faint and bigger transcript, which probably is caused by the presence of Tos17 (4.1 kb) in the transcript. For osjar1-3, we detected a transcript smaller than in the wild type. With probe N2, we could only detect an OsJAR1 mRNA signal in the wild type (Figure 3G), thus in osjar1-3 the Tos17 insertion causes premature termination of transcription of OsJAR1 mRNA. To summarize, we conclude that transcription of the OsJAR1 is disrupted in osjar1- 2 and osjar1-3 and that no active OsJAR1 protein can be formed.
Rescue of osjar1 phenotype by overexpression of OsJAR1
To confirm that the osjar1 phenotype was indeed due to Tos17 insertions in OsJAR1, we set up a complementation test by overexpressing a wild type copy of the OsJAR1 cDNA under control of the rice Act1 promoter (construct ProActin::OsJAR1) in stably transformed rice plants. When osjar1-2 was transformed with construct ProActin::OsJAR1, the anther indehiscence phenotype was rescued in the resulting plants. Three ProActin::OsJAR1 transgenic lines with a single T-DNA insertion were selected and they showed a normal floret opening and closure phenotype. Normal seed set was produced as well (Figure 4B). Northern blot analysis of the three single T-DNA transgenic lines showed expression of wild type OsJAR1 mRNA (Figure 4C). Because the transgenic lines were
Figure 4. Rescue of the osjar1-2 phenotype by transgenic overexpression of OsJAR1.A. Effect of MeJA on the induction of floret opening of Actin::OsJAR1 transgenic lines (□ and ×) and wild type (△and ◇). Each data point represents the mean (with S.D.) of ten replicates. Concentrations of MeJA were 0 mM (□ and △) and 0.4 mM (◇ and ×). B. Morphology of grains (from left to right) from wild type, osjar1-2 and Actin::OsJAR1 plants, respectively. C. Northern blot analysis of OsJAR1 in osjar1-2, wild type and Actin::OsJAR1 transgenic lines. WT, the wild type; A173, A61 and A143 represent three independent single T-DNA insertion lines with construct ProActin::OsJAR1. Hybridization with 17S rRNA was to check the loading of total RNA.
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made via a tissue culture-based protocol that in principle can activate Tos17 elements, Southern blot analysis was performed to verify any Tos17 activity from OsJAR1. However, the Tos17 element was still inserted at the same position in the transgenic osjar1-2/ProActin::OsJAR1 lines tested (data not shown).
OsJAR1 mRNA is expressed in floral and vegetative organs
The temporal and spatial expression pattern of the OsJAR1 gene was analyzed in further detail using an OsJAR1 promoter-β-glucuronidase (ProOsJAR1::GUS) fusion construct. For this, a 4.5 kb promoter fragment was translationally coupled to the GUS reporter gene and introduced in Nipponbare rice via Agrobacterium-mediated transformation. In total, 25 independent transgenic plants were made expressing construct ProOsJAR1::GUS. GUS reporter gene activity was detected in leaf blade, sheath, root, floret and stem of mature plants (Figure 5A-5D). In the fibrous root system, ProOsJAR1::GUS was predominantly expressed in young adventitious root rather than older adventitious roots (Figure 5A). Strongest GUS activity was found on the base of new lateral roots and no GUS activity occurred at the tip of the root (Figure 5A). Analysis of cross section of ProOsJAR1::GUS main root showed that ProOsJAR1::GUS is more abundant in vascular cylinder than in cortex cells (exodermis, sclerenchymatous layer cells, cortical parenchyma cells and endodermis) and epidermis (Figure 5O). In vascular cylinder, no GUS activity was detected in protoxylem and metaxylem (mxI and mxII); strong GUS activity was detected in pericycle, parenchyma cells and primary phloem; primary phloem has the strongest ProOsJAR1::GUS expression (Figure 5P). ProOsJAR1::GUS show similar expression patterns in mature leaf blade, leaf sheath and stem. That is, strongest expression was detected in the vascular bundle of these three tissues (Figure 5E-5L). In addition to the vascular bundle, weak GUS activity was detected in mesophyll cells and bulliform cell, but not in the sclerenchymatous layer cells in mature leaf blade (Figure 5F). In mature leaf sheath and stem, GUS activity was detected in the parenchyma cells between two vascular bundles (Figure 5H, 5J, and 5K). In the vascular bundle of these three tissues, GUS activity was detected in bundle sheath, protoxylem parenchyma cells, metaxylem parenchyma cells, sieve-tube cells, companion cells and metaphloem parenchyma cells as well (Figure 5L-5N).
Companion cells and metaphloem parenchyma cells has the strongest ProOsJAR1::GUS expression.
No GUS activity was detected in the late metaxylem (mxII) and weak GUS activity was detected in early metaxylem (mxI) (Figure 5M-5N).
The ProOsJAR1::GUS construct showed a dynamic expression pattern in floret during development. In the early developmental stage (one week before floret opening), GUS activity was detected in glumes only, but not in stamen, pistil and lodicules (Figure 6A). When florets get older (2~4 days before anther dehiscence), GUS activity was detected in the lodicules, the upper part of ovary and the connective of anthers adjacent to the filament (Figure 6B). Up to one or two days before floret opening till the end of floret opening, obvious GUS activity was detected in all parts of the connective tissue of the anthers (Figure 6C, 6D and 6E). Furthermore, the part of filament close to the anther showed ProOsJAR1::GUS expression during anther dehiscence (Figure 6D) and GUS activity was found in the whole filament at the end of anther dehiscence (Figure 6E). The expression of ProOsJAR1::GUS construct in stamen becomes more obvious by comparing anthers from different developmental stages together (Figure 6O). By checking cross sections of anthers from different developmental stages, we realized that the majority of anthers in the early or middle
87 Figure 5. Histochemical localization of ProOsJAR1::GUS in root, leaf sheath and stem of transgenic rice plants. A. Adventitious roots. B. Internodes. C. Segment of leaf blade. D. Segment of leaf sheath. E.
Transverse section through the region of main vein of a mature leaf blade. F. Transverse section through the region of lateral large vascular bundle of a mature leaf blade. G. Close-up of main vascular bundle in (E). H.
Transverse section through the first internode showing the internode wall. I. Close-up of large vascular bundle in (H). J. Transverse section through a leaf sheath. K. Transverse section through the midrib region of a leaf sheath. L. Close-up of the large vascular bundle in (K). M, N. Details of the large vascular bundle in (L). O.
Transverse section through a primary root 2 mm from the tip. P. Details of central vascular cylinder in (O). bf, bulliform cell; bs, bundle sheath; cc, companion cells; cpc, cortical parenchyma cells ,ed, endodermis; ep, epidermis; ex, exodermis; mc, mesophyll cells; mpp, metaphloem parenchyma cells; mvb, main vascular bundle; mxI, early metaxylem; mxII, late metaxylem; mxp, metaxylem parenchyma cells; pc, pericycle; pp, primary phloem; pxl, protoxylem lacuna; pxp, protoxylem parenchyma cells; px, protoxylem; sc, sieve-tube cells; sl, sclerenchymatous layer cells; vb, vascular bundle; vc, vascular cylinder; vpc, parenchyma cells in vascular bundle. Bar (A) = 0.2 mm; Bars (B-D) = 1 mm; Bars (E, K) = 100 μm; Bars (F, H, Q) = 50 μm; Bars (G, I, L) = 25 μm; Bar (J) = 250 μm; Bars (M, N, P) = 15 μm.
microspore stage had no ProOsJAR1::GUS expression (Figure 6F), but there is still a minority of anthers in the early or middle microspore stage showing weak GUS activity in the vascular bundle cells (VBC) of the anther connective tissue (Figure 6K). Obvious GUS activity was found in the
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Figure 6. Localization of ProOsJAR1::GUS expression in florets of transgenic rice plants. A-E. Floret and stamen at one week before floret opening (A), the stage of 2~4 days before anther dehiscence (B), the early stage of anther dehiscence (C), the middle stage of anther dehiscence (D) and the end of anther dehiscence (E).
F-K. Transverse sections of anther in different developmental stages, which is microspore stage (F, K), the stage of 2~4 days before anther dehiscence (G), the stage of 1~2 days before anther dehiscence (H), the early stage of anther dehiscence (I) and the end of anther dehiscence (J). L Details of the vascular bundle in the stage just before anther dehiscence. M-N. In situ hybridization of OsJAR1 transcript in the transverse sections across the region of the connective tissue of the anther obtained from a wild type floret just before dehiscence.
(M) Antisense probe. (N) Sense probe. O. Stamen in different developmental stages. an, anther; ft, filament; pa, palea; lo, lodicules; V, vascular bundle. Bars (A-E) = 1 mm; Bars (F-J) = 50 μm; Bars (K-O) = 25 μm.
VBC from the late microspore stage onwards (Figure 6G). Compared to the late microspore stage, a more intense GUS activity was detected in the VBC during the pollen mature stage (Figure 6H).
During the early stage of floret opening, GUS activity was detected not only in the VBC, but also in the septum (Figure 6I). Till the end of anther dehiscence, GUS activity was detected in the vascular bundle of the anther connective tissue, septum and anther wall as well (Figure 6J). Strong expression of construct ProOsJAR1::GUS in the VBC of the anther connective tissue is consistent with the results obtained from in situ hybridization of cross sections of anthers with an anti-sense OsJAR1 RNA probe (Figure 6M). A sense probe did not show any positive signal in the VBC of the anther connective tissue (Figure 6N).
osjar1 has normal pollen viability and pistil perceptiveness
Plants with the osjar1-2 (30 plants tested) and osjar1-3 (10 plants tested) mutations showed 9.1%
fertility (ranging from 7.5% to 14.5%) whereas wild type plants showed 98% fertility. Fertility was measured as the ratio between filled grains and total number of florets. In order to find out which defects in flower organs resulted in the observed lower fertility of osjar1, crossing assays and pollen viability tests were performed. After crossing of 25 osjar1-2 pistils with mature pollen from control Nipponbare plants, 25 seeds were obtained. All the progeny of the crossed lines showed no
89 Figure 7. KI-I2 staining and in vitro germination of rice pollen. A-C. KI-I2 staining of mature pollen grains from osjar1-2 (A) and osjar1-3 (B) and wild type (C). D-F. In vitro germination of mature pollen grains from osjar1-2 (D) and osjar1-3 (E) and wild type (F). Bars = 0.1 mm
Figure 8. Inhibition of root growth by MeJA in wild type and osjar1. A and C. Root growth of wild type (A) and osjar1-2 (C) in medium in the absence of MeJA. B and D. Root growth of wild type (B) and osjar1-2 (D) in medium with 10 μM MeJA. Photographs were taken with same magnification seven days after germination.
E and F. Inhibition of root growth of osjar1-2 and wild type plants in response with MeJA. Each data point represents the mean (with S.D.) of at least ten replicates. Concentration of MeJA were 0 μM (◇), 1 μM (□), 10 μM (△), 25 μM (×), 50 μM (＊) and 100 μM (○), respectively.
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differences in phenotype compared to control plants, indicating that the stigma of osjar1-2 is able to accept pollen for fertilization. Furthermore, in vitro pollen germination and iodine stainings were carried out to check viability of mature osjar1-2 pollen. Results show that 95.6% (478/500) of wild type pollen and 93% (465/500) pollen from osjar1-2 were stained with iodine (Figure 7A-7C). In the pollen germination assay, 90.8% (590/650) and 86.8% (564/650) of the pollen from control and osjar1-2 plants, respectively, germinated two hours after incubation in a pollen germination medium (Figure 7D-7E). Essentially, the same results were obtained for mutant osjar1-3, which suggested that pollen of osjar1-2 and osjar1-3 are able to pollinate the stigma and that osjar1 mutants have a normal, functioning and receptive pistil.
osjar1-2 shows reduced root growth inhibition on MeJA
It has been reported that MeJA inhibits root growth in Arabidopsis (Staswick et al., 1992). In contrast to wild type, the Arabidopsis jar1-1 loss-of-function mutant exhibits moderate insensitivity to MeJA in a root growth inhibition assay (Staswick et al., 1992). We examined whether root growth of rice is sensitive too to MeJA by germinating rice seeds on media containing various concentrations of MeJA. As shown in Figure 8A-8D, seven days after germination, control seedlings showed 45% and 70% inhibition of primary root growth on medium with 1 μM and 10 μM MeJA, respectively. On higher concentrations MeJA (25, 50 or 100 μM), a more serious inhibition of primary root growth occurred (Figure 8E). In contrast to the obvious inhibition of root growth of control plants, osjar1-2 roots exhibited considerable MeJA-resistance and showed only 7% and 30%
growth inhibition on media with 1 μM and 10 μM MeJA, respectively. Addition of 100 μM MeJA resulted in 50% root growth inhibition in osjar1-2 seedlings (Figure 8F).
osjar1 mutants show reduced resistance to the blast fungus Magnaporthe oryzae
JA is implicated in disease resistance mechanisms in plants (Berger, 2002; O'Donnell et al., 2003;
Jones and Dangl, 2006; Qiu et al., 2007) and several defects in disease resistance were found in the Arabidopsis jar1 mutant (Staswick et al., 1998; Ferrari et al., 2003; Traw et al., 2003). We investigated the possible role of the OsJAR1 gene in resistance to the fungus M. oryzae which is the causative agent of rice blast disease. When inoculated with the avirulent strain CL367, mutants osjar1-2 and osjar1-3 showed larger necrotic lesions than wild type plants (Figure 9A). Consistently, ProActin::OsJAR1 lines overexpressing the OsJAR1 gene in cultivar Nipponbare showed reduced susceptibility to the virulent isolate FR13 (Figure 9B) which normally is pathogenic to Nipponbare rice. This suggests that the OsJAR1 gene is required for blast fungus resistance. We further investigated regulation of OsJAR1 by following the expression of the ProOsJAR1::GUS construct in infected plants. Surprisingly, GUS activity decreased around the lesions triggered by the avirulent isolate CL367 (Figure 9C). In virulent-infected plants (isolate FR13), GUS activity was also reduced with the notable exception of the immediate surroundings of the lesions (Figure 9C). The results show that OsJAR1 is tightly controlled by infection with M. oryzae with contrasting effects depending on the distance to the infection site.
MeJA and JA-mediated induction of floret opening in osjar1 is impaired
It has been reported that the opening of florets in rice can be induced by different concentrations of MeJA (Zeng et al., 1999). We checked if opening of florets from osjar1-2 were also inducible by
91 Figure 9. OsJAR1 is a positive regulator of resistance towards rice blast disease. A. The insertion mutant osjar1-3 shows reduced resistance (enhanced necrosis) to the avirulent strain M. oryzae CL367. Similar results were obtained with osjar1-2. B. The over-expression line ProActin::OsJAR1 shows reduced susceptibility (grayish lesions) to the virulent strain M. oryzae FR13. Similar results were obtained with five independent transgenic lines. C. Expression of construct ProOsJAR1::GUS is regulated by infection with M. oryzae. (a) Non-infected plants only treated with gelatin used for resuspending M. oryzae spores. GUS activity is detected in the entire leaf blade. (b) Plants inoculated with the avirulent strain M. oryzae CL367. The brown spots are typical for a resistance response. GUS activity is reduced around the necrotic lesions. (c) Plants inoculated with the virulent strain M. oryzae FR13. The non-stained around the infection sites are surrounded by intense GUS staining represent typical susceptible lesions after infection with M. oryzae. In contrast, GUS activity is reduced in tissues distant from the infection sites. All experiments were repeated three times on four independent transgenic lines.
Figure 10. Effect of MeJA on induction of floret opening in wild type and osjar1-2. A. Control plants. B.
osjar1-2 mutant. Each data point represents the mean (with S.D.) of at least ten replicates. Panicles were treated with 0, 0.04, 0.4 and 4 mM MeJA, respectively. Concentration of MeJA were 0 μM (◇), 1 μM (□), 10 μM (△), 25 μM (×), 50 μM (＊) and 100 μM (○), respectively.
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MeJA. Prior to treatments with MeJA, any already flowering florets were removed. This experiment was taken three hours in advance of the predicted opening time of florets from control Nipponbare plants. Indeed, floret opening in control plants was inducible by MeJA. The first opening of florets on control plants occurred in 17 to 25 minutes after MeJA treatment, depending on the different concentrations of MeJA. A higher concentration of MeJA was inducing more floret opening per panicle (Figure 10A). Surprisingly, florets from osjar1-2 plants were still closed, even 90 minutes after treatment in various tested MeJA concentrations (Figure 10B). Essentially, similar results were obtained with osjar1-3. In summary, the results show that the MeJA signaling pathway involved in floret opening is defective in the osjar1 mutants. Furthermore, by treating transgenic ProActin::OsJAR1 lines with MeJA during anthesis, responsiveness of floret opening and closure by MeJA was fully restored, which confirmed that the MeJA-mediated pathway for floret opening was rescued by overexpression of the wild type OsJAR1 cDNA in osjar1-2 (Figure 4A). We also used JA to treat osjar1-2 and wild type flowers. Similar results were observed in which the induction of floret opening in wild type was impaired in osjar1 too (data not shown).
Rescue of the phenotype of osjar1-2 by exogenous JA treatment
One of the main characteristics of the phenotype of osjar1 is that the lodicules keep on swelling even several days after opening of the florets started. We checked if JA treatment could rescue the phenotype of the osjar1 lodicule. Ten flowers from two osjar1-2 plants at 1 DAF were treated with JA and ten other osjar1-2 flowers were treated with DMSO as control treatment. The treatment was started at 9 am and no obvious change was observed throughout the daytime till 9 pm. However, all JA-treated lodicules were found withered (Figure 11B) and the flowers closed 24 hours after treatment, whereas the control osjar1-2 lodicules did not (Figure 11C) and florets stayed opened.
OsJAR1 is responsive to wounding and exogenous JA/MeJA
Riemann et al. (2008) described that the expression of OsJAR1 to responsive to exogenous application of MeJA within 45 minutes. Since osjar1 exhibits insensitivity towards MeJA treatment in different assays with a variety of timeframes of treatments, we further investigated the temporal responsiveness of OsJAR1 expression by exogenous JA and MeJA application. Northern blot analysis was used to monitor OsJAR1 expression in segments of wild type seedling leaves cut into two centimeter pieces and incubated with JA and MeJA respectively. DMSO and water were used as controls. Initially, we checked the effects on OsJAR1 expression within two hours of incubation.
Accumulation of OsJAR1 mRNA started within half an hour in all treatments including the control, which indicates that wounding also triggers OsJAR1 expression. However, MeJA application extra induced OsJAR1 expression at 0.5 hr (Figure 12). For JA, no obvious differences compared to the controls were discovered. To further study the temporal regulation of OsJAR1, we analyzed the expression profile at time points ranging from 3 to 24 hr after starting the experiment. Extra inductions were both found with JA and MeJA treatment at 3hr. After that, the mRNA level OsJAR1 of all treatments decreased at 6 hr to the original level but increased again to a comparatively higher level than zero from 12 to 24 hr. Thus, the expression may be controlled by the circadian rhythm too.
93 Figure 11. Rescue of the osjar1 flower phenotype by JA treatment. Ten lodicules of osjar1-2 flowers at 1 DAF were treated with JA by pipetting 3 µl of 5mM JA onto the lodicules very carefully. Ten osjar1-2 flowers were treated with 4% DMSO as controls. A. osjar1-2 flower two days after flowering start. B. osjar1-2 flower 24 hours after treated with JA. C. osjar1-2 flower 24 hours after treated with 4% DMSO.
Figure 12. OsJAR1 is responsive to wound and application with JA/MeJA. Northern blot analysis of leaf segments from three-week-old seedlings after treatment with 100 μM JA, 0.4 mM MeJA, 1% DMSO and water controls in a time-dependent manner under continuous light. Total RNA was extracted at the time points (in hours) indicated above the lanes. The lane indicated with 0 indicates the start of the experiment.
OsJAR1 encodes an enzyme catalyzing the formation of JA-amino acid conjugates
OsJAR1 is predicted to encode a protein of 581 amino acids that belongs to the plant-specific GH3 family. Database searches and phylogenetic analysis showed that OsJAR1 is the most closest to NaJAR4 and AtJAR1 from tobacco and Arabidopsis respectively (Wang et al., 2007). Alignment of the deduced OsJAR1 amino acid sequence showed that it has ~61% identity (79% similarity) and
~67% identity (83% similarity) with AtJAR1 and NaJAR4 respectively, which implicated that OsJAR1 might have similar functions like AtJAR1 and NaJAR4. Both AtJAR1 and NaJAR4 encode a JA-Ile synthetase capable of conjugating JA to isoleucine (Staswick and Tiryaki, 2004; Kang et al., 2006). To study if OsJAR1 protein has this function too, recombinant protein was expressed as a glutathione S-transferase (GST) fusion in E. coli. After purification, the putative activity of the recombinant GST-OsJAR1 protein was assayed in independent enzymatic reactions with all 20 different L-amino acids. Reactions without GST-OsJAR1 protein were set up as negative controls.
After analysis by thin-layer chromatography (TLC), eight amino acids were found conjugated to (±)- JA in presence of GST-OsJAR1. Figure 13 shows that the putative conjugates for JA-Ala, JA-Cys,
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Figure 13. Conjugation of eight different amino acids with JA by recombinant GST-OsJAR1 protein.
Reactions with JA and each amino acid (three letter abbreviations) were analyzed by TLC and stained for JA with vanillin dye. (+) and (-) represent with and without recombinant protein respectively.
Figure 14. Variation of in vitro synthesized JA-AA conjugates and measurement of JA-AA content in leaves from wild-type and osjar1-2 plants. A. LC-MS of enzymatic synthesized JA-AA. B and C UPLC-MS measurement of JA-AA content in flag leaves from wild type (B) and osjar1-2 (C) plants, five hours after 10mM JA treatment. D and E UPLC-MS measurement of JA-AA content in flag leaves from wild-type (D) and osjar1-2 (E).
95 JA-Ile, JA-Leu, JA-Met, JA-Phe, JA-Trp and JA-Val all have reduced mobility compared to JA.
There are also obvious differences in intensity of the JA spots when either of the eight conjugates were formed, which indicates the reduction of free JA because of the conversion into conjugated forms.
Six of the in vitro conjugation products of JA with amino acids (Ile, Leu, Val, Met, Phe, Trp) were further analysed using ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). For each conjugate, five or six peaks of variable intensity were observed in the corresponding chromatograms (Figure 14A). MS/MS fragmentation of the individual peaks produced very similar fragmentation spectra, strongly suggesting that the peaks belong to (diastereo) isomeric compounds. So far the chromatographic separation of jasmonate-N-amino acid isomers has only been described for chiral HPLC columns and has not been possible with classical C18 reversed phase columns (Kramell et al., 1999). With UPLC a much better separation of closely eluting compounds is achieved. Without comparison with authentic standards it is very difficult to assign the correct chemical structure to each of the isomers. The synthetic (±) JA that was used is a mixture of a number of (stereo) isomers. According to the UPLC-MS/MS chromatogram, the main constituent at 4.85 min is a mixture of the (3R,7R) and (3S,7S)-2-Z-pentenyl stereo isomers (approximately 89% according to LC-MS/MS analysis). The (3R,7S) and (3S,7R)-2-Z-pentenyl stereo-isomers (9%) elute at 4.95 min, while the minor peak (2%) at 5.00 min could be a mixture of the (3R,7R) and (3S,7S)-2-E-pentenyl stereo isomers (Holbrook et al., 1997). The relative concentration of the JA-AA isomers produced will be a function of the starting concentration and of the substrate specificity of the recombinant GST-OsJAR1 protein towards each of the JA stereo- isomers. For all JA-AA conjugates isomer 3 (assigned on the basis of elution order in the chromatograms) is the major metabolite formed. It is likely that this is the (3R, 7R)-jasmonate-N- amino acid isomer (Vick and Zimmerman, 1984; Mueller and Brodschelm, 1994; Holbrook et al., 1997).
Reduced levels of JA-AA conjugates in leaves from osjar1-2 plants
Based on the observation that recombinant OsJAR1 protein is able to catalyze synthesis of eight different JA-AA conjugates, leaves from wild type and osjar1-2 plants were analyzed for JA-AA content using UPLC-MS/MS. In the first experiment we analyzed leaf materials treated with 10mM JA in order to confirm if this treatment would affect levels of JA-AA conjugates. In the MRM chromatograms of extracts collected from wild type leaves treated with (±) JA, four or five isomeric jasmonate conjugates were detected that correspond to Ile, Leu, Val, Met and Phe, conjugation of JA to Trp could not be detected (Figure 14B). There are clear differences between extracts obtained from wild type and osjar1-2 (Figure 14B and C). Isomer 3 of JA-Ile and JA-Leu dominate in the wild type extract. Significant amounts of JA-Val isomer 3 and isomer 1 of JA-Leu and JA-Val are present as well, while isomer 4 is absent for all AAs. However, in the same leaf tissue from osjar1-2 the overall concentration of jasmonate-N-amino acid conjugates is significantly lower than in wild type (aproximately 5-fold). This is mainly due to the fact that the reduced concentration of isomer 3 of JA-Ile, JA-Leu and JA-Val in osjar1-2 compared to wild type (25-fold reduction of JA-Ile and 10- fold reduction of JA-Leu and JA-Val). The other isomers are present more or less in the same quantities as in wild type, with isomer 1 of JA-Leu and JA-Val as predominant conjugates. In the chromatograms of extracts obtained from untreated wild type and osjar1-2 leaves, the same isomers
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of JA-Ile, JA-Leu, JA-Val and JA-Phe conjugates were detected as in the JA-treated leaf extracts, but in concentrations more than 100-fold reduced (Figure 14D and E). Thus, JA treatment induces accumulation of JA-AA conjugates in rice flag leaves. The distribution of isomers and relative abundance of JA-AAs in untreated wild type leaves is very similar to that of JA-treated wild type. In both samples, isomer 3 of JA-Ile and JA-Leu are the most abundant JA-conjugates. The conjugate profile for the extract from osjar1-2 bears a closer resemblance to that of wild type rather than that of JA-treated osjar1-2. The high preference for isomer 1 JA-AA conjugates in leaves from osjar1-2, treated with JA, is not found in untreated osjar1-2. JA-Ile and JA-Val isomer 1 together with JA-Ile isomer 5 are most abundant in extracts from osjar1-2 leaves. On average the levels of JA-AA conjugates are three times lower in osjar1-2 extracts.
Discussion
JA is a growth regulator and has been shown to control a wide range of processes in plants, ranging from responses to biotic and abiotic stresses to various developmental and environmental response processes (Ellis and Turner, 2001; Berger, 2002; Mei et al., 2006; Acosta et al., 2009). However, the molecular mechanisms involved in these processes are yet poorly understood. Previously, the OsJAR1 gene was reported to be regulated by exogenous MeJA and two different photoreceptors, phytochrome A (phyA) and phytochrome B (phyB). The corresponding mutants, osjar1-2 and osjar1-3 are characterized by a very obvious floret phenotype with open husks and also includes low-fertility and malformed seeds (Riemann et al., 2008). In this Chapter, we studied the phenotype of osjar1 and the molecular and physiological actions of OsJAR1 in more detail and show that this protein has a biochemical function as an enzyme conjugating amino acids to JA resulting in active hormones which is confirmed by the finding that JA conjugates are lower in osjar1-2 plants. The lowered fertility was shown to be caused by a strongly reduced anther dehiscence and not be any obvious defect of the pistil since these can be pollinated normally. The osjar1 pollen itself could also be ruled out as the cause of reduced fertility since they were shown to be perfectly viable in pollen assays. We tested for disease susceptibility of plants misexpressing OsJAR1 and also showed a role for this gene in resistance towards the rice blast fungus Magnaporthe oryzae. Finally, we postulate that the lowered levels of the JA conjugates cause the open husk phenotype of the two osjar1 alleles studied here.
The genetic basis of the phenotype of osjar1-2 and osjar1-3
In rice, opening and closure of the florets results from swelling of lodicules which pushes away the palea from the lemma and subsequent withering, respectively (Matsuo and Hoshikawa, 1993).
Normally, rice florets close again in less than 90 minutes after opening because the lodicules are withering quickly after swelling. However, in both Tos17 alleles for osjar1 that we studied, the lodicules remained swollen even five days after floret opening started. Consequently, movement of the palea and lemma and thus, closure of the flower are impaired. Other phenotypical aspects of osjar1 are that the seeds are narrower and more elongated and that the style remains attached. The fertility is much lower than in wild type Nipponbare plants grown under the same conditions. This phenotype is observed in both alleles of osjar1 which both segregate as a recessive allele. On northern blots we observed abnormal osjar1 transcripts for both alleles. For osjar1-2 in line NC2728,
97 expression was much reduced and the length of the band correlated with the insertion of the 4.5 kb Tos17 retrotransposon in exon 3. For osjar1-3 in line ND0051, we observed that the osjar1 mRNA was shorter which may be caused by premature termination of transcription or incorrect splicing. We also identified a third Tos17 allele in line NE8577, but this line has no observable phenotype. This Tos17 insert is in the fourth intron and usually such Tos17 inserts are spliced out together with the intron without having too many effects on transcription. Strong support for the hypothesis that the two mutant lines with the osjar1 phenotype are caused by Tos17 insertions in OsJAR1 and thereby impairing the function of the gene product is by the experiment in which we retransformed osjar1-2 with an actin promoter-driven OsJAR1 overexpression construct. Five independent transgenic lines were verified for overexpression of OsJAR1 and showed complementation of the osjar1 phenotype.
The seeds of these transgenic plants could not be distinguished from those from wild type Nipponbare plants and the induction of opening of the florets by MeJA is restored as well without any other observable effects on the phenotype Because Tos17 is activated by tissue culture we confirmed by Southern blotting that Tos17 is still present in all the transgenic lines.
OsJAR1 has functions as a JA-AA synthetase
OsJAR1 was confirmed to encode a JA-AA synthetase by TLC and LC-MS/MS assays. For this, recombinant OsJAR1 protein was purified from E. coli and was shown to catalyze the formation of JA-Phe, JA-Ile, JA-Leu, JA-Met, JA-Trp, JA-Val, JA-Cys and JA-Ala in in vitro reactions analyzed with TLC assays. All eight amino acids that were conjugated by OsJAR1 to JA belong to the so- called non-polar group (Alberts et al., 2008), indicating OsJAR1 has a certain substrate preference.
The identity of JA-Ile, isolated from TLC plates, was further confirmed by LC-MS/MS analysis.
Furthermore, UPLC-MS/MS analysis was used to determine JA-AA levels in wild type and osjar1 plants. In total, five conjugates were confirmed that corresponded to Ile, Leu, Val, Met and Phe of which JA-Leu and JA-Ile are the dominant forms. Unlike the jar1 mutants in Arabidopsis which only show an obvious decrease of JA-Ile (Staswick and Tiryaki, 2004), in osjar1-2 plants we observed significant reduction in levels of five out of six measured JA-AA conjugates and in particular the level of JA-Ile is 25-fold lower. Also JA-Leu and JA-Val levels are strongly affected with a ten-fold decrease. It is therefore very likely that the low levels of JA-Ile and JA-Leu or JA- Val are responsible for the observed phenotype of the open-staying flowers and delayed anther dehiscence. There are a number of other studies that implicate a function of JA in these processes.
The Arabidopsis mutants fad3-2 fad7-2 fad8 (McConn and Browse, 1996) and opr3 (Stintzi and Browse, 2000) have defects in JA biosynthesis and all have lowered levels of JA. In these mutants, flower organs initially develop normally, but like with osjar1-2 and osjar1-3, anther locules do not dehisce at the right time of flower opening. A major difference is that the filaments do not elongate normally and pollen grains are inviable whereas these are unaffected in osjar1-2 and osjar1-3. An experiment involving treatment of leaves from osjar1-2 and wild type plants with JA further verified the role of OsJAR1 as a JA-AA synthetase in vivo. UPLC-MS/MS results showed that this treatment resulted in a strong accumulation of JA-AA conjugates and that in wild type leaves, isomer 3 of JA- Ile was 25-fold higher than in osjar1-2 and also JA-Leu and JA-Val were 10-fold higher. Although the accumulation of JA-AA conjugates was much less in osjar1-2, the level of JA-AA conjugates also increased considerably compared to the untreated leaves, indicating there are also other, redundant JA-AA synthetase expressed and active. The induction of the level of isomer 1 JA-Leu
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and JA-Val in osjar1-2 and wild type was not so different, which further demonstrated the activity of other JA-AA synthetases. Taken together, from the results we can conclude that OsJAR1 has substrate preference and is mainly responsible for the conjugation of isomer 3 JA-AA molecules. In the experiment of flower opening induced by JA treatment, the induction was found impaired in osjar1-2 and osjar1-3. Because the most apparent difference in the level of JA-AA conjugates between wild type and osjar1-2 was found in isomer 3 JA-Ile and JA-Leu, we speculate that these two are the most essential conjugates involved in flower opening and closure and anther dehiscence.
Treatment of osjar1-2 lodicules with exogenous JA can rescue the lodicule phenotype in osjar1-2 yet needs a comparatively long period (24 hours), indicating that other JA-AA isomers or even other JA-related pathways may also be involved in flower opening but it could also be that it is difficult for JA to get into the lodicule.
Function of JA-AA conjugates in plants
Experiments in Arabidopsis show that JA-Ile is an important signaling molecule in the COI1- mediated JA-response, and acts by promoting the physical interaction between COI1 and JAZ1 proteins and initiates SCFCOI1 dependent proteasome degradation of JAZ repressor proteins. In turn, this results in release of the transcription factor MYC2 (and probably other proteins) from the MYC2-JAZ complex, and subsequent activation of transcriptional activation of JA response target genes (Farmer, 2007; Thines et al., 2007). Since the JA-Ile content in leaves of osjar1 plants is much lower, it is very well likely that this will have effects on downstream genes involved in lodicule swelling and withering, anther dehiscence and seed-shape. Effects on gene expression also has been shown for the Arabidopsis opr3 mutant where transcriptome studies of the stamen (Mandaokar et al., 2006) showed groups of genes that are specifically induced or repressed by JA, amongst which are many transcription factors and transporters. Recently, Staswick (2009) reported that JA-Trp functions as an endogenous auxin inhibitor in Arabidopsis and that exogenous JA-Trp application caused agravitropic root growth in seedlings. The biological functions of other JA-AA conjugates are still unclear. It is known that the homeostasis of an active hormone is rather important for normal plant development and defence responses and that the homeostasis of active auxin is maintained by IAA-AA hydrolases and IAA-amido synthetases (Staswick et al., 2005; Schuller and Ludwig-Muller, 2006). It is very well possible that JA-AA conjugates especially JA-Ile and JA-Trp and associated biosynthesis genes including OsJAR1 and others have an important function in the homeostasis of active jasmonates in planta. If so, there are probably yet unknown JA-AA hydrolases acting together with the OsJAR1 protein in the conversion of active jasmonates into inactive JA-AA conjugates, resulting in the homeostasis of active jasmonates in plants.
The lower fertility of osjar1 mutants is caused by a reduction of anther dehiscence
Although osjar1 mutant has a 7.5-14.5% lower seedset because of flower sterility, it showed normal pistil receptiveness in crossing experiments. Furthermore, KI-I2 stainings in in vitro pollen germination assays showed that nothing is wrong with viability of the pollen themselves. However, we found that in fact anther dehiscence is much reduced in osjar1 plants. While normally anthers are dehiscing and withering after pollen release, most of the osjar1 anthers remain closed or only partially opened although they seem to develop normally and are completely and normally filled with pollen grains. Dehiscence of the anthers is the result of a multistep process that starts with
