Investigation of the molecular mechanisms causing Cold induced sterility in rice

A report for the Rural Industries Research and Development Corporation by

Nijat Imin Dr Jeremy J Weinman

Professor Barry G Rolfe

April 2004 RIRDC Publication No 04/002 RIRDC Project No ANU-29A

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© 2004 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0 642 58712 4 ISSN 1440-6845 “Investigation of the molecular mechanisms causing cold induced sterility in rice” Publication No. 04/002 Project No. ANU-29A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquires concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details Professor Barry G. Rolfe Genomic Interactions Group, Research School of Biological Sciences, ANU. PO Box 475, Canberra, ACT 2601 Phone: 02 6249 4054 Fax: 02 6249 0754 E-mail: rolfe@rsbs.anu.edu.au Dr Jeremy Weinman Genomic Interactions Group, Research School of Biological Sciences, ANU. PO Box 475, Canberra, ACT 2601 Phone: 02 6249 5071 Fax: 02 6249 0754 E-mail: weinman@rsbs.anu.edu.au Nijat Imin Genomic Interactions Group, Research School of Biological Sciences, ANU. PO Box 475, Canberra, ACT 2601 Phone: 02 6249 2441 Fax: 02 6249 0754 E-mail: imin@rsbs.anu.edu.au In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4539 Fax: 02 6272 5877 E-mail: rirdc@rirdc.gov.au. Website: http://www.rirdc.gov.au Published in April 2004 Printed on environmentally friendly paper by Canprint

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Foreword The growing of rice in irrigated regions of NSW is a major agricultural enterprise, which relies on sophisticated farming techniques to produce some of the highest grain yields in the world. However, some major environmental factors limit the expansion and indeed the future viability of this industry. These factors are the availability of water, and the occurrence of cool mid-season temperatures that give rise to considerable pollen sterility and consequent lowered grain yields. Cool mid-season temperatures are commonly combated by a procedure of deep flooding in the irrigation bays which serves to act as a thermal damper. This practice is becoming increasingly difficult to maintain, as environmental and political pressures seek to divert more of the water from the Snowy Mountain Scheme catchment into restoring flows in the Snowy River. This limits the amount of water available to rice producers. To assist efforts to breed more cool tolerant varieties and thus lessen the dependence of the industry on water, it is highly desirable to understand the mechanisms underlying the sterility. As a part of a concerted effort to understand the mechanisms that cause mid-season pollen sterility, the RIRDC has funded a number of projects that investigate causes and possible treatments for cool-temperature induced pollen sterility. More recently, much of this research has come under the broader umbrella of the Cooperative Research Centre for Sustainable Rice Production, of which the RIRDC is a major partner. Aspects of this research project will be continued through funding from the CRC. This report details fundamental research that characterises proteins the levels of which are substantially changed in anther tissue as a result of growth under cool mid-season temperatures. This has enabled the identification of cellular processes and protein markers that are affected by these temperatures. This project was funded from industry revenue that is matched by funds provided by the Australian Government. This report, a new addition to RIRDC’s diverse range of over 1000 research publications, forms part of our Rice R&D program, which aims to improve the profitability and sustainability of the Australian rice industry. Most of our publications are available for viewing, downloading or purchasing online through our website:

downloads at www.rirdc.gov.au/reports/Index.htm purchases at www.rirdc.gov.au/pub/cat/contents.html

Peter Core Managing Director Rural Industries Research and Development Corporation

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Acknowledgements The authors would like to acknowledge the valued assistance and skills of the Biomolecular Resource Facility at the ANU and the Australian Proteome Analysis Facility at Macquarrie University for N-terminal sequencing and Peptide Mass Fingerprinting, respectively. We also thank the Applied Biosystems Division of the Perkin Elmer Corporation for assistance with sequencing by tandem mass spectrometry. Robert Williams and his physiology team at Yanco Agricultural Institute and the plant culture staff at the Research School of Biological Science are gratefully acknowledged for their dedication in the cultivation of rice plants. Jan Elliott of the RSBS is thanked for her expertise with generating immune responses in rabbits. The Cooperative Research Centre for Sustainable Rice Production is acknowledged for its assistance in bringing together Australian scientists studying the phenomena of mid-season sterility in rice for yearly workshops. Finally, we are extremely grateful for the significant funding for this project, in addition to that provided by the RIRDC, which has been provided by the Research School of Biological Sciences at the Australian National University.

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Contents Foreword iii Acknowledgments iv Executive Summary vi 1. Introduction 1 1.1 Cytological changes and the role of pollen 1 1.2 Biochemical changes 1 1.3 A need for greater understanding 1 1.4 Proteomics: a study of the whole 2

Objectives 3

2. Methodology 4 2.1 Plant Materials, Growth and Sampling 4 2.2 Extraction of rice anther proteins 6 2.3 2-DE 7

2.4 Silver staining, colloidal coomassie staining, Sypro Ruby 7 fluorescent staining and image analysis

2.5 Western blotting and N-terminal sequencing 8 2.6 Peptide Mass Fingerprinting and tandem mass spectrometry analysis 8 2.7 Techniques for antibody production and probing 9 3. Results 10 3.1 Verification of sterility caused by cold-treatment 10

3.2 Determination of the correlation between Auricle Distance and the 11 developmental stage of the pollen

3.3 Cold treated plants show cytological abnormalities after cold treatment 13 3.4 Proteome maps of rice anthers 13 3.5 Detection of cold responsive proteins 24

3.6 N-terminal microsequencing, de novo sequencing and 26 Peptide Mass Fingerprinting

3.7 Use of cold responsive anther proteins for antibody production 28 4. Discussion 29 4.1 Explanation of the approach 29 4.2 Rice anther proteome map established 29 4.3 Most of the anther proteins are unaltered by cold treatment 29

4.4 The relation between the functions of cold responsive proteins and their 31 potential roles in microspore development and their response to cold

4.5 Antibody production and immunohistochemistry 33 5. Implications 34 6. Recommendations 35 7. References 36

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Executive Summary Reproductive cold damage is the main environmental limitation to yields in the NSW rice industry, causing an average loss of approximately 2 t/ha over the last 10 years. In extreme years this can cost the rice industry over $60M. The mechanisms by which cold temperatures cause pollen sterility are poorly understood and this lack of knowledge needed to be addressed to accelerate the rate at which resistance to cold-temperature sterility can be introduced into current commercial cultivars. Past research has been unable to identify the causes of the sterility. Recent technological advances in Proteomics offer the ability to discover what causes sterility and provide the breeding program with an understanding of what traits to incorporate for resistance to cold-temperature serility.

A major advantage of the Proteomic approach is the ability to separate and visualise the entire cellular complement of proteins, at a specific developmental stage or from a specific organ or tissue. By subtracting the elements of this display from those produced from untreated or control tissues the proteins that are differentially accumulated between the two treatments can be identified. This technology presented itself as a strong experimental approach to be used in order to produce a greater understanding of the mechanisms that cause mid-season cold-temperature induced sterility. We focused on the identification of the gene products that are affected by the first few days of the process of sterility triggered by cold temperatures in rice anthers. The experimental procedure was growth of rice and 1, 2 and 4 day cold-temperature treatment at the cold-sensitive pollen stage, micro-dissecting the anthers, analysing them for their total protein complement, and characterising proteins altered in amount by the cold-treatment. This was done for both the cold-sensitive Australian cultivar Doongara and the relatively cold-tolerant Hungarian cultivar HSC-55. Proteome analysis established the first two-dimensional anther protein maps of rice cultivars Doongara and HSC-55. Over 4,000 anther proteins at the young microspore stage were reproducibly resolved, representing up to 20% of the estimated total genomic output. The most cold-responsive proteins observed after plants were treated with a 12oC cold treatment were selected and analysed to predict their function. After comparison of many independent experiments, more than 50 Doongara anther proteins were found to be responsive to cold stress. Among them, one protein is newly induced, 47 protein spots are up regulated and 9 protein spots down-regulated in anthers after cold-treatment. From these, 9 anther proteins have now been identified with confidence. They include a lipid transfer protein down regulated after cold treatment and a translation initiation factor eIF-5a which is newly induced after cold treatment. The levels of these proteins did not vary in panicle samples nor in the anthers of the relatively cold-tolerant cultivar HSC-55 in response to cold treatment. This implicates these proteins as playing a role in the process of cold induced sterility and suggests mechanisms involved in cold damage.

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Figure 1. Scheme used in the project. Anthers from the top spikelets of the terminal 3 branches were harvested, dissected out and proteins extracted for Proteome analysis. Proteomes of normally grown plants were compared with those of cold-treated ones to reveal cold-responsive proteins. These were then identified. Three cold-responsive proteins were selected to be bulked up for use in raising antibodies against these proteins. While no immune response has been obtained, the very small amounts of protein available make this task quite difficult. Recently, however, samples sent to the USA have been analysed by state of the arts instrumentation to yield primary sequence from within the proteins. This sequence can now be used to generate large amounts of artificially produced sub-sections of the protein and this can then be used to elicit a good antibody response. This work will continue for the next year under funding from the CRC for Sustainable Rice Production. Antibodies to these cold responsive Doongara proteins that were identified in this project can be used as molecular markers for identification of more cold-tolerant cellular metabolism, and be used to help breed increased cold-tolerance into existing high yield cold susceptible cultivars.

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Introduction Rice (Oryza sativa) evolved in tropical and subtropical areas and as a result it has characteristics of being vulnerable to cold weather. Specifically, the combination of the low temperature sensitivity and a maximal sensitivity to these temperatures at precise stages of pollen microspore development (Satake, 1976), makes cool mid-season temperatures the major environmental limitation (other than soil and water) for growing rice in temperate regions. During the life cycle of rice, pollen microspore development at the booting stage is known to be most susceptible to cool weather damage (Nishiyama 1995). Cold damage at this reproductive stage of rice, causes limitation to the yields of temperate grown rice around the world, and yield reductions of up to 40% (Angus and Lewin 1991; Nishiyama 1984). In Australia it has been estimated that cold damage causes an average loss of 2 t/ha, and in extreme years as in 1996 it was estimated that yield losses in the crop were 3.5 t/ha. The main cause of this damage is pollen sterility resulting from low temperature damage at the early stage of microspore development. The most sensitive stage to the various forms of environmental stress, including cold damage, is just after meiosis, that is, tetrad to early microspore phase - or the young microspore stage (Satake and Hayase 1970). 1.1 Cytological changes and the role of pollen In cold-treated rice plants, both cytological and histological abnormalities are greater in the anthers than in the pistil or other organs of the flower and the cold damage can be rescued by artificial pollination with healthy pollen (Terao, 1940, Sakai, 1943). In this process of cold damage, anthers become smaller, cells in the tapetal layer in the anthers which surrounds and is responsible for transferring nutrients to the developing pollen undergo hypertrophy and eventual breakdown. As a result, the normal development of pollen grains does not occur, and the pollen grains contain little or no starch and are functionally sterile (Nishiyama 1984, 1995). 1.2 Biochemical changes In anthers from cold-treated rice plants, anther respiration decreases, sucrose accumulates in anthers, protein levels drop and amino acid composition also changes including a large decrease in proline and increase in asparagine (Ito, 1970). Recently, Kawaguchi et al. (1996) found that a novel tetrasaccharide Ara2Gal2, which closely resembled the glycan chain of arabinogalactan-protein, decreases after cold-treatment. Because the ß-1,3-glucanase, or callose hydrolyses the callose wall and release tetrads (Steiglitz, 1973), Tsuchiya (1995) suggested that premature dissolution of the callose wall may cause male sterility. Saini et al. (1996) found a decrease in invertase activity in water stressed rice anthers at the meiosis stage, suggesting that a disturbance in carbohydrate metabolism may be involved in male sterility subjected to water stress. Kitashiba (1999) demonstrated partial male sterility in transgenic tobacco carrying an antisense gene for alternative oxidase under the control of a tapetum-specific promoter suggesting an important role of alternative oxidase in pollen development. It is also known that for many rice varieties, top dressing with N-fertiliser exacerbates the problem of cold-induced sterility. Again, the reason for this observation is unknown. 1.3 A need for greater understanding In spite of much recent research on the subject, the underlying mechanisms that cause cold-induced male sterility are poorly understood. In particular, it is not known if the tapetal abnormalities, the tapetal hypertrophy or the biochemical changes seen are the causes of the processes which lead to pollen sterility or their consequences. In order to identify the earliest processes that are affected by cold-treatments, we focused on the identification of the anther gene products that are initially affected during the process of sterility triggered by cold temperatures in rice. To identify which gene products are involved in this process of cold-induced male sterility, we have used proteome analysis technique coupled with high-resolution two-dimensional electrophoresis (2-DE) to investigate the effect of cold-treatments on the rice anther proteome.

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1.4 Proteomics: a study of the whole Proteomics is the study of the proteome, the total protein output encoded by the genome. It deals with the analysis of whole genomes at the functional protein level by describing the protein complement expressed by the genome of an organism, tissue or differentiated cell (Wilkins et al., 1997). Proteome studies have developed from and are dependent upon the core technology of two-dimensional polyacrylamide gel electrophoresis for the separation of 1000s of proteins from complex protein mixtures. Significant advances in bioinformatics and techniques for protein/peptide identification have enabled high sensitivity and throughput in protein analysis. This has made proteome analysis a primary tool for characterising gene expression and regulation in complex biological systems (Guerreiro et al., 1997). In essence, Proteomics is the study of protein properties (expression level, post-translational modification, interactions etc) on a large scale to obtain a global, integrated view of disease processes, cellular processes, cell responses to environmental stresses, and cell networks at the protein level. This field has rapidly grown in importance because of three basic questions that need to be addressed by all the genome-sequencing projects: (a) what are the functions and characteristics of all the gene products - the proteins? (b) a poor correlation (generally below 0.5) between mRNA abundance and the amount of the corresponding functional protein present in the cell raises questions on a nucleic acid based approach alone, and (c) the recent recognition that one gene can give rise to many protein products through different post-translation modifications of the same original gene product now necessitates the analysis of proteins. A major advantage of 2-DE is the ability to separate and visualise the entire cellular complement of proteins, at a specific developmental stage or from a specific organ or tissue. By subtracting the elements of this display from those produced from untreated or control tissues the proteins that are differentially accumulated between the two treatments can be identified. This technology presented itself as a strong experimental approach to be used in order to produce a greater understanding of the mechanisms that cause mid-season cold-temperature induced sterility.

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Objectives The originally stated objectives of the research project were: “Developing a clearer understanding of the mechanisms that cause cold-induced sterility will enable rice breeders to target specific physiological traits for selection in their ongoing program to develop varieties with lower yield losses due to cold-induced sterility.” The project was designed to use an approach that was not constrained by a limited number of biochemical assays, nor by only describing changes seen under the microscope. This Proteome based approach was conceived to gain a clearer understanding of the cellular components of anthers that respond to cold-temperature treatments, and to use this knowledge to predict what processes in the anther are being affected. An additional aim, facilitated by this approach, was to develop molecular markers that will enable rice breeders to target specific physiological traits for selection of more cold tolerant rice varieties. The project also contributes to the understanding of the molecular basis of male gametogenesis and its response to environmental stresses.

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Methodology 2.1 Plant Materials, Growth and Sampling 2.1.1 Plant Materials Rice (Oryza sativa) cultivar Doongara, one of the most cold sensitive commercially used Australian long grain varieties, and the relatively cold tolerant recent accession Hungarian cultivar HSC-55, were supplied by Robert Williams, Yanco Agricultural Institute of New South Wales. Soil used in all the experiments was sourced from the Yanco Agricultural Institute, and originated from fields in the Murrumbidgee Irrigation area. 2.1.2 Determination of critical stage Japanese Research had previously determined that rice is most sensitive to cold temperatures at the early young microspore stage (Satake, 1976). In order to maximise the degree of cold damage to the anthers in the plants we were growing, we needed to know when this developmental stage occurred in order to determine when to initiate our cold treatments. This determination had to be through a correlative non-destructive method, as the developing anthers are within the leaf sheath at this stage, and determining the stage directly would kill the plant. Thus we used the auricle distance (AD, see Figure 2), the distance between the auricle of the flag-leaf (last leaf) and that of the immediate lower leaf, as a non-destructive tool for the identification of the stage of development of the rice panicle and pollen microspore. The developmental stage is defined in Table 1 below.

Table 1. Classification of Male Microsporogenesis of Doongara

Stages Microspore Description Auricle Distance (mm) 1 Pollen mother cell Relatively large (40mm x 40mm). <-20 2 Tetrad Young microspores (10mm x 10mm) in

tetrad form in callose wall. -18 to -10

3 Early microspore No germpore on microspore. No vacuoles. -10 to 0

4 Middle microspore Has multiple vacuoles. Size of vacuole is small.

-5 to + 50

5 Late microspore Has multiple vacuoles. Size of vacuoles relatively large.

+40 to +100

6 One-nucleate Clear exine formation, starch formed in the pollen.

+90 to >+100

We have determined the correlation between AD and microspore development stages for the terminal five spikelets of the top three panicle branches. In particular, an AD of -18 to -10 mm for Doongara and -100 to -80 mm for HSC-55 corresponds with the tetrad to early microspore stage of microspore development (see results). We used this measurement to determine the most temperature sensitive stage for the start of cold treatment, and this also allowed us to determine other developmental stages without sacrificing the growing plant.

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Figure 2. Auricle Distance (AD) is measured as the distance between the ligule of the flag leaf and the penultimate leaf. AD is expressed in mm. When the flag leaf has not emerged from the leaf sheath, the AD value will be negative.

2.1.3 Growth conditions, cold treatment and anther sampling During this project we continually refined the growth system used. Three different controlled environment facilities were used for rice growth, cold treatment and anther sampling: (1) A Samples (Yanco samples). In the first year, we grew rice plants (Doongara) at the Yanco Agricultural Institute, NSW. Plants were grown under flood in a glasshouse under the following conditions: 24ºC/20ºC (day/night) for untreated plants and 20ºC/12ºC (day/night) for cold treated plants, 70% relative humidity (RH) and natural day length. Nitrogen (150kg/ha) was applied as urea to 4-week old seedlings. Plants were grown until the microspore development stage and anthers from the top spikelets at the critical stage were harvested as described below (2.2), except kept on dry ice (-30ºC) until storage back in Canberra in a –80ºC freezer. Due to limiting controlled environment facilities for plant growth and cold treatment at the Yanco Agricultural Institute, we needed to establish plant growth facilities in the Research School of Biological Sciences in Canberra to provide continuous cycles of plant growth and cold treatment. The samples from Yanco were used for method development and reference only. (2) B Samples (First RSBS samples). Rice plants (Doongara) were grown one plant to a pot under flood in a greenhouse under the following conditions until the tetrad stage of microspore development: 30ºC/20ºC (day/night), 70% RH and natural day length. Nitrogen (150 kg/ha) was applied as urea to 4-week old seedlings. Cold treatments were performed by moving the plants that were at the early microspore stage (see below for stage determination) into an identical chamber kept at 12ºC (day and night), 70% RH, with a photon flux intensity of 330 µM.m-2.s-1. and a day/night cycle of 12/12 h. While these samples were collected at precise AD stage, the effect of varying day length through the year introduced some variability in the correlation of AD to pollen developmental stage between batches of rice. (3) C Samples (Precisely controlled samples). Rice plants (Doongara and HSC-55) were grown in individual pots under flood in a greenhouse under the following conditions until panicle initiation: 30ºC/20ºC (day/night), 70% RH and natural day length. Around the panicle initiation, the plants were transferred into a growth chamber under the following conditions; 30ºC/20ºC (day/night), 70% RH and a day/night cycle of 12/12 h, at an average photon flux intensity of 330 µM.m-2.s-1. Nitrogen (150kg/ha) was applied as urea to 4-week old seedlings. For Doongara plants that were given additional nitrogen, nitrogen (150 kg/ha) was applied as urea at the PI (panicle initiation) stage. Cold treatment was performed by moving individual plants that were at the early microspore stage (see below for stage determination), into an identical chamber kept at 12ºC, 70% RH, with the same illumination and day/night cycle of the control condition. These samples were sampled at precise developmental stages and show tighter

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correlation of AD to developmental stage due to the absence, after panicle initiation, of varying daylight periods between batches of rice grown throughout the year. The last controlled environment provided our most controlled sets of conditions for plant growing, cold treating and sampling. We have been growing 4 tubs (width 36 cm, length 52 cm, and height 30 cm) of rice plants (24 T7 pots in each tub, one plant in each pot) every 4 weeks. Using T7 pots (height 15 cm, diameter 8.3 cm), we were able to limit the number of tillers below 3 which is critical to control variation of the samples due to different tiller sizes. Cold-temperature treatment severely delays the rate of microspore development. Consequently, in order to be able to compare anthers from cold-treated plants with normally grown anthers at an identical stage of development, we used anthers from normally grown plants that had the same AD as the treated plants after the completion of their cold-treatment. 2.1.4 Microscopic methods for anther analysis Anthers at the early stage of pollen development (tetrad to early microspore stage, at which anthers 0.8-1.0 mm long) were removed from the upper 15 spikelets of top three branches of immature panicles with precision forceps and scalpels under a dissection microscope. Anthers were stained either by heating with acetocarmine solution (4% carmine in 45% acetic acid, boiled for one hour under reflux, then filtered when cooled), or incubation with DAPI (4,6-diamidino-2-phenylindole, 0.5 mg/L in 50% ethanol) or with toluidine blue (pH 4.4; O’Brien and McCully, 1981) solution without heating. They were either pre-fixed with FAA solution (50% ethanol, 5% acetic acid) if they were to be stored prior to staining and microscopy, or they were stained directly if microscopy proceeded immediately. DAPI stained samples were visualised under UV light. Alternatively, for embedding and sectioning, anthers were fixed at least overnight in FAA solution, dehydrated at 4ºC in increasing concentrations of ethanol (70, 80, 90 and 96% for 1 h each) and infiltrated with Historesin (Leica Instruments, Heidelberg, Germany) at 4ºC on a rotating wheel overnight. Sections of 10 µm were cut on a Reichert Jung Ultra Microtome using freshly cut glass knives. Sections were transferred to glass slides and stretched onto the slide surface, then stained with a drop of toluidine blue on top of each section. The slides were placed on a warming plate at 40ºC for 15 mins after rinsing with water several times. Colour micrographs of the stained anthers were taken with a Nikon FX35 camera mounted onto a Nikon SMZ-10 stereo microscope using Kodak Ektachrome 100 ASA, EPY 64T or Agfachrome RSX100 colour reversal films. 2.2 Extraction of rice anther proteins Anthers at the early stage of pollen development (tetrad to early microspore stage, at which anthers are about 0.8-1 mm long) were removed from the spikelets of top three branches of immature panicles with precision forceps and scalpels under a dissection microscope. These were put into eppendorf tubes, snap frozen in liquid nitrogen, and kept at –80−C in an ultra cold freezer until use. Before extraction they were pooled together to make about 100 mg samples. To obtain enough material for a single 100 mg extraction, approximately 5,000 anthers had to be harvested from the panicles of over 55 rice plants containing anthers at the same stage of development. The extraction method was that of Damerval et al. (1986), with some modifications. Anthers were ground in liquid nitrogen for about 10 mins until they had become a very fine powder and were then suspended in 5 mL of 10% trichloroacetic acid (TCA) in acetone containing 0.07% DTT (dithiothreitol), and sonicated with the tubes on dry ice using a probe sonicator (MSE 100, Thomas Optical and Scientific Co Ltd.) in 5 x 20 second bursts at 7 microns (peak amplitude) with 60 s intervals between bursts. After standing for 1 hr at -20ºC, samples were centrifuged at 35,000 g for 15 mins. The precipitates were washed twice with pre-chilled acetone containing 0.07% DTT, standing for 30 mins or overnight at -20ºC before each centrifugation at 12,000 g for 15 mins, and then were dried by lyophilisation (Flexi-Dry MP, FTS Systems Inc., Stone Ridge, NY) for 5 minutes. The resultant powder was suspended in 250 mL of urea lysis buffer consisting of 9M urea, 4% w/v 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), 1% w/v DTT, 1% v/v carrier ampholytes pH 3-10 (BioRad), 35 mM Tris. The samples were lysed by treatment in an ultrasonic water bath (Transonic 460, John Morris Scientific, NSW) for 20 mins with 30 s vortexing intervals for each 1.5 mins treatment and centrifuged at 13,000 g for 5 mins at room temperature. The protein

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concentration of the supernatant was determined by the Bradford assay (Bio-Rad, Hurcules, CA, USA) with bovine serum albumin fraction V as the standard. The supernatants were stored in aliquots at –80oC or directly loaded for isoelectric focusing. Each independent experiment has been done at least twice unless specified. Anther pellet extraction was performed as follows: pellets remaining in the tubes after the above extraction had been washed twice with urea lysis buffer (see above) with ultrasonic solubilisation and precipitation by centrifugation at 13,000 g for 15 min. These remaining pellets were each re-extracted with 400 µL SDS lysis buffer (2% SDS, 1% DDT, 10% glycerol and 62.5 mM Tris-HCl pH 6.8) by treating in an ultrasonic waterbath (see 2.2 above) and boiling for 5 mins. Then, a 10 times volume of pre-chilled acetone was added to each sample, left at –20oC for at least 30 mins or overnight. After centrifugation at 13,000g for 15 mins, the pellets were lyophilised. 50 µL of 1% SDS lysis buffer (1% SDS, 1% DDT, and 62.5 mM Tris-HCl pH 8.8) were added to each sample and boiled for 5 mins. Then 200 µL of thiourea lysis buffer consisting of 7 M urea, 2 M thiourea (Sigma), 4% CHAPS 4% w/v 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), 1% w/v DTT, 1% v/v carrier ampholytes pH 3-10 (BioRad), 35 mM Tris was added to each sample after the sample cooled down to room temperature. They were solubilised in an ultrasonic water-bath for 20 mins as described above. 2.3 2-DE 2-DE was carried out in a horizontal electrophoresis system, Multiphor II (Pharmacia, Uppsala, Sweden) as previously described (Görg et al. 1988; Guerreiro et al. 1997; Görg et al. 1998) with minor modifications. For the first dimension, IPG gel strips (180 mm x 3 mm, pH 4-7, 6-11 and 3-10L; Pharmacia-Biotechnology, Uppsala, Sweden) were rehydrated for at least 6 h in a solution containing 8 M urea, 0.5% w/v CHAPS, 0.2% w/v DTT, 0.52% w/v carrier ampholytes 3-10 and 0.006% w/v bromophenol blue. Sample application was at the anodic end with a cup-loading of 100 µg (0.5-2 mg for preparative gels) total protein and run with the Immobiline DryStrip kit in a Multiphor II electrophoresis unit (Pharmacia-Biotechnology) for 200 kVh (100 kVh for 3-10L 18 cm IPG strips) at 20ºC. After the first dimensional run, the IPG gel strips were sealed in plastic and frozen at -80ºC, or incubated at room temperature to equilibrate the strips in loading buffer prior to separation in the second dimension. Equilibration involved two 10 min washes. The first was in 15 mL of an equilibration solution A containing 6 M urea, 0.05 M Tris-HCl, pH 8.8, 30% glycerol (ICN, Costa Mesa, CA), 2% w/v SDS (Genomic Solutions, Chelmsford, MA, USA) and 2% w/v DTT. The second equilibration wash was in 15 mL of equilibration solution B, made up as for the solution A but in which 4% w/v iodoacetamide and 0.02% bromophenol blue (2.5% for preparative gels) were added to the solution in place of the DTT. The equilibrated IPG gel strips were then placed onto Pharmacia ExcelGel SDS gels (T=12-14%) for second dimension SDS-polyacrylamide gel electrophoresis on a Multiphor II unit. 2.4 Silver staining, colloidal coomassie staining, Sypro Ruby fluorescent staining and image analysis Proteins on analytical 2-DE gels were visualised by silver staining essentially as described by Rabilloud (1992) but with the addition of 2.5 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid], hemi-sodium salt (HEPES; Research Organic Inc., Cleveland, OH) buffer to the silver nitrate solution. Colloidal coomassie staining was performed by staining the gels in 0.1% G-250 solution (17% ammonium sulfate, 3% phosphoric acid, 34% methanol and 0.1% Coomassie G-250) (Neuhoff, et al. 1988; Anderson, et al. 1991) and destained in deionised (Milli-Q; Millipore) water. Stained gels were digitised at a 600 dots per inch resolution using a UMAX PS-2400x lamp scanner fitted with a UTA-II transparency adapter under Photoshop 5.0 (Adobe, Mountain View, CA). For Sypro Ruby fluorescent staining, gels were fixed with 40% methanol, 10% acetic acid for 30 mins after the completion of second dimensional run, then stained with Sypro Ruby gel stain (Genomic Solutions Inc., Ann Arbor, MI, USA) overnight. No destaining was needed. The plastic gel support (GelBond) had to be removed prior to scanning with a FluoroImager (Molecular Dynamics, Sunnyvale, CA, USA) without a filter under ImageQuant (version 5.1) program. Spot detection, spot measurement and gel-to-gel protein spot matching was performed with Melanie II 2-

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DE software (GeneBio, Geneva, Switzerland). The apparent molecular masses of the proteins were determined by co-electrophoresis with the following standard proteins (Pharmacia-Biotechnology): phosphorylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and lactalbumin (14.4 kDa). 2.5 Western blotting and N-terminal sequencing Semi-dry electrophoretic blotting onto polyvinylidene difuoride (PVDF) membranes (Bio-Rad) was performed in a Pharmacia apparatus using a transfer buffer containing 10 mM 3-[cycohexylamino]-propanesulfonic acid, pH 11.0, in 10% v/v methanol and 0.01% SDS as previously described (Matsudaira, 1987). Electro-transfer was performed at 0.8 mA/cm2 for 90 min. Membranes were stained with 0.1% w/v Coomassie brilliant blue R-250 (Bio-Rad) in a 40% methanol solution for 5 min and then destained in a 50% methanol, 5% acetic acid solution for 10 min. The membranes were then washed in water and allowed to air dry then stored, sealed in plastic at -20ºC. Selected spots were excised from dried membranes for N-terminal amino sequencing. N-terminal protein sequencing was carried out at the Biomolecular Resource Facility of the ANU. This was done using Edman degradation chemistry on a PROCISE-HT sequencer system or on a PROCISE-CLC (both from Perkin-Elmer Applied Bio-systems, Foster City, CA, USA) for very low abundance proteins. For amino acid sequence homology comparison, N-terminal protein sequences were used to search non-redundant protein and nucleic acid databases (SWISS-PTOR, PIR, TREMBL, GenPept and TIGR rice databases) using the FASTA (http://www2.ebi.ac.uk/fasta3, http://www-nbrf.georgetown.edu), BLAST (http://www.ncbi.nlm.nih.gov/blast) and WU-BLAST (http://www.tigr.org/cgi-bin/BlastSearch/blast_tgi.cgi?) programs. 2.6 Peptide Mass Fingerprinting and tandem mass spectrometry analysis Proteins on analytical 2-DE gels were visualised by colloidal coomassie staining as mentioned above. Selected spots were excised from polyacrylamide gels and shipped to the Australian Proteome Analysis Facility (APAF) where they underwent an in gel 16 hour tryptic digest at 37ºC. The resulting peptides were extracted from the gel with a 50% (v/v) acetonitrile, 1% (v/v) triflouroacetic acid (TFA) solution. A 1 mL aliquot was spotted onto a sample plate with 1 mL of matrix (α-cyano-4-hydroxcinnamic acid, 8 mg/mL in 50% v/v TFA) and allowed to air dry. Matrix assisted laser desorption ionisation (MALDI) mass spectrometry was performed at APAF with a Micromass ToFSpec 2E Time of Flight Mass Spectrometer to generate peptide mass fingerprints. Peptide mass fingerprinting (PMF) homology comparisons were done with the PeptIdent program available from ExPASy (www.expasy.ch), searching against Swiss-Prot and TrEMBL databases. Isoelectric point and molecular mass windows of ± 1.0 unit and ± 20% respectively were used. All peptide masses were assumed as monoisotopic and [M+H]+ (protonated molecular ions). Peptides with cysteines were searched assuming that these residues had been reduced and alkylated by iodoacetamide (forms carboxyamidomethyl cysteine, Cys_CAM). Searches were conducted using a mass accuracy of ± 0.5 Da. Searches against GenPept databases were also done with the MS-Fit program available at the UCSF Mass Spectrometry Facility (http://prospector.ucsf.edu/ucsfhtml3.2/msfit.htm). Matches assigned to the peptide mass fingerprint were based on multiple criteria including the species the protein matched to, the number of PMF matches, the number of missed cleavages, the score, % sequence coverage and compatibility of isoelectric and molecular mass of the analysed and database protein. For tandem mass spectometry analysis, the selected spots were digested at APAF as described above and then were shipped to the USA and analysed on a PE SCIEX QSTAR Hybrid LC/MS/MS Quadrupole TOF System in Foster City, CA. For protein sequence homology comparisons, full or partial amino acid sequences generated by decomposition analysis were used to search non-redundant protein database and nucleic acid databases (SWISS-PTOR, PIR, TREMBL, GenPept and TIGR rice databases) using the FASTA (http://www2.ebi.ac.uk/fasta3, http://www-nbrf.georgetown.edu), BLAST (http://www.ncbi.nlm.nih.gov/blast) and WU-BLAST (http://www.tigr.org/cgi-bin/BlastSearch/blast_tgi.cgi?) programs as described above.

9

2.7 Techniques for antibody production and probing Anther proteins of interest were excised from colloidal coomassie stained 2-DE gels and pooled together (from 4-10 gels each) to provide an amount sufficient to trigger an immune response. These gel spots were ground in liquid nitrogen until they had become a very fine powder and were then suspended in PBS solution (Phosphate buffered saline, 0.15 M NaCl, 0.1 M Na2HPO4/NaH2PO4, pH 7.4). All the materials and solutions used in the experiment were pre-sterilised except the adjuvants. 500 µL of soluble proteins in PBS were mixed well with Freud’s complete adjuvant (Freud’s incomplete adjuvant was used for boosting; both from Pierce, Rockford, Illinios, U.S.A.) using a three-way stop-cock (Sigma). Alternatively, antigens in PBS solution were used directly. These were injected at four subcutaneous sites of 2-3 month old New Zealand white rabbits. Boosts were spaced at 3-4 week intervals using half the starting amount of the antigens. Pre-immune bleeding was performed by taking 2 mL of blood from the ear of the rabbits before the first injection. Test bleedings were performed 6-10 days after the second, third and fourth boosting. All blood samples were kept overnight at 4oC and spun at 5,000g for 10 minutes. Serum (supernatant) was collected and stored at –80oC until use. For Antibody western probing, anther protein samples were extracted with SDS lysis buffer (2% SDS, 1% DDT, 10% glycerol and 62.5 mM Tris-HCl pH 6.8) as described in section 2.2. They were run on Tris-glycine pre-cast mini gels (4-20% T) (Novex, San Diego, CA) according to the manufacturer’s guidelines. Then they were blotted onto immun-blot PVDF membranes (Bio-Rad) as described above (section 2.5). Membranes were blocked with a blocking agent made from 7.5% skim milk, 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.05% Tween 20 for 1 hour. Then they were rinsed once with TBS, placed into primary antibodies diluted 1:200-10,000 times in TBS solution (20 mM Tris-HCl pH 7.5, 150 mM NaCl) for 1 hour or longer at room temperature. The membranes were washed with TBS with 0.05% Tween 20 three times for 5-10 mins each and then reacted with horseradish peroxidase conjugated secondary antibody (diluted 1,000-10,000) for 30 mins. After three washes with TBS containing 0.05% Tween 20 for 5-10 mins each, signal detection was performed with a enhanced chemiluminescence detection kit (ECL, Pharmacia) according to the manufacturer’s guidelines.

10

Results The cold sensitive Australian commercial cultivar Doongara was extensively studied in this project. The relatively cold tolerant Hungarian cultivar HSC-55 was subsequently studied in order to examine difference between the responses of the two cultivars. 3.1 Verification of sterility caused by cold-treatment We used cold treatment and One of the primary requirements in the design of the project was to make use of a cold-treatment that would result in sterility and subsequent lowered grain yields, thus being a treatment that would mimic the effects seen in the field. This requirement was brought home strongly, when growth to the mature seed stage of 5 day cold-treated rice plants grown by the A Sample method in Yanco revealed that negligible sterility had resulted. This is one of the reasons that growth was moved to Canberra and a 12/12 degree C day/night regime used. By growing rice plants under these conditions (B and C samples), considerable sterility was observed in Doongara plants which had had a cold-treatment commenced at the young microspore stage (see below). Figure 3 shows an example of an experiment in which sterility is determined in plants grown by the C Sample method with varying lengths of cold-treatment. The top 15 spikelets of the cold sensitive cultivar Doongara plants showed about 4% sterility (scored as an empty seed husk at head fill) without cold treatment and one day cold treatment at a constant 12oC initiated at the young microspore stage did not have much effect on the plant, only increasing the measured sterility by 2%. However, both two days and four days of cold treatment caused about 33% and 84% sterility respectively. It is known that the low temperature damage at young microspore stage can be exacerbated by nitrogen application (Amano and Moriwaki, 1984; Heenan, 1984). In our system, the sterility of 4 days cold treated top spikelets after nitrogen application at the panicle initiation stage was 90%, that is, 6% more sterility than the sterility of the plants to which no nitrogen is applied at the panicle initiation stage. In contrast, the cultivar HSC-55 shows considerably greater tolerance to cold-treatment having a sterility of 24% after 4 days of cold-treatment and 1% sterility without cold-treatment (Figure 3).

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Figure 3. Sterility of the spikelets from the top three branches with and without cold treatment at 12ºC for different days at the early stage of microspore development in cold sensitive cultivar Doongara and relatively cold tolerant cultivar HSC-55. All data is for Doongara unless indicated as HSC which stands for Hungarian cultivar HSC-55. 4D(N@PI): 4 days cold treatment of the plants to which nitrogen fertiliser applied at panicle initiation stage. 3.2 Determination of the correlation between Auricle Distance and the developmental stage of the pollen A second major requirement in the development of the experimental system was to be able to maximise the amount of homogeneous sample that could be collected. We wished to minimise experimental variation due to sampling from different stages of anther development. To minimise this effect it was important not to sample anthers from all spikelets in a growing panicle, as these spikelets undergo a staggered developmental program. We chose to use a method similar to that used previously by others in the field (Heenan, 1984) and sample only the terminal 5 spikelets of the top 3 branches in a panicle, as these have similar developmental timing. As the effect of cold-temperature is also known to affect the developing pollen grains individually, rather than all or none of them in an anther being affected, it was important to maximise the affect of cold on the pollen grains in the anthers that were to be collected. Previous Japanese and Australian work (Satake, 1976; (Heenan, 1984) had identified the tetrad formation/early young microspore stage as the developmental stage at which, if a cold-treatment is initiated, the greatest numbers of pollen grains will be affected and will thus lead to maximal sterility. Consequently, we used a measure of Auricle Distance (AD) to permit the developmental stage of the anthers growing within the leaf sheath to be predicted. Figure 4 shows how we have been able to maximise the number of the top 15 spikelets, which are at the tetrad formation - early young microspore stage by using specific AD’s to predict developmental stage. AD is the standard parameter to estimate the panicle development not the microspore development. However, we used AD to estimate microspore development as a non-destructive tool after determined its correlation with microspore development. Note that this correlation may change depending on different cultivar used and different environmental conditions (e.g. Radiation, soil, temperature etc) the plants grow.

Figure 3. Sterility of the top spikelets after cold treatment (%)

0

20

40

60

80

100

control 1Day 2 Days 4 Days 4D(N@PI) HSC control HSC 4D

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Figure 4. Auricle Distance & Microspore Development

0%

20%

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-30 -20 -18 -15 -12 -10 -5 0 10

Auricle Distance (mm)

Perc

enta

ge in

eac

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age PMC

TetradEMMM

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Figure 4. Correlation between the auricle distance and the microspore development of the spikelets of the top three branches of the Doongara panicle. Horizontal axis shows AD (auricle distance) of the plants tested, while the vertical axis shows percentage of the spikelets in each stage of microspore development among the spikelets from the top three branches. Results were from the average of at least three independent experiments. Tetrad: tetrad stage; PMC: pollen mother cell stage; EM: early microspore stage; MM: middle microspore stage. Figure 5. Cross sections of Doongara anthers stained with toluidine blue at the early and middle stage of microspore development. A, B, C, D & E are the cross sections of the untreated, 2 days at 20C/12C, 2 days at 12C/12C, 4 days at 12C/12C and 6 days at 12C/12C respectively. F, G, H, I and J are showing enlarged loculi of A, B, C, D and E respectively. Swelled tapetal cells indicated with arrows. 3.3 Cold treated plants show cytological abnormalities after cold treatment As previous research has demonstrated cytological abnormalities in the tapetal cells due to cold treatment, we also examined the affect of the cold treatment we were using on the anthers harvested from the upper 15 spikelets. Under the microscope, cross-sections of cold-treated anthers showed abnormal tapetum and microspore development in the cold sensitive cultivar Doongara. There is a severe swelling of tapetal cells known as tapetal hypertrophy observed after cold treatment. Microspores also showed abnormal development (Figure 5). 3.4 Proteome maps of rice anthers A complete proteome for any organism or any typical organ is highly complex. Functional proteins have different characteristics and properties. Very hydrophobic proteins, in particular membrane proteins that are known to play many important roles, are under-represented on 2-DE protein maps due to their low solubility. Resolving very basic and very high or low molecular weight proteins presents a continuing challenge for the technology. Low abundance proteins (1-2000 copies per cell), especially regulatory molecules, are not easily detected on the 2-DE gels. To overcome these problems, different solubilisation, fractionation and narrow range IPG strips have been applied: 1. Standard solubilisation by CHAPS-UREA for total proteins and increased solubility by SDS-CHAPS-UREA for very hydrophobic proteins used in the panicle and anther proteome generation. Although SDS (sodium dodecyle sulfate) is one of the strongest detergent ever known, but as being an ionic detergent, it is not compatible with isoelectric focusing in the first dimension. However, by diluting the SDS extractions with other non-ionic detergents, SDS can be used and enhances solubilisation of hydrophobic proteins to a great degree. 2. For eukaryotic organisms, membrane proteins may represent up to 30% of the genome encoding capacity (Walli and von Heijne, 1998). To analyse the effect of cold treatment on membrane proteins, panicle plasma membrane proteins were isolated and solubilised with SDS-CHAPS-UREA prior to 2-DE analysis. 3. A range of IPG strips (all 18cm long) was used to achieve maximum resolution of proteins over most biological pH ranges. They were pH 3-10L (linear) for overall expression, pH 4-7L for the best coverage, pH 6-9L and pH 6-11L for basic proteins and pH 5-6L to expand a crowded section of the pH 4-7 Proteome. These various methods have been used in the proteome analysis and comparison of cold-treated and control samples. The main aim of these trials is to display as much proteins as possible to compare their response to cold treatment. This is a very important step in identifying most of the gene products potentially involved in the process of cold-induced sterility in rice. 3.4.1 Panicle We initially tested panicle samples for optimising and standardising the protein extraction and solubilisation procedures. Figure 6. The resultant Proteome maps are the first that have been produced for developing rice panicles.

15

16

17

18

19

20

21

22

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Figure 6. 2-DE map of Doongara panicle proteins. Isoelectric focusing in the first dimension was on IPG strips with a linear gradient ranging from pH 4-7 (18 cm) and loaded with 100 ug of total cellular protein. For the second dimension 12-14 %T SDS-PAGE gels were used. Molecular weight is indicated with MW (kDa). Proteins were visualised by silver staining. Figure 7A. 2-DE map of untreated Doongara anther proteins at the early stage of microspore development. Isoelectric focusing in the first dimension was on IPG strips with a linear gradient ranging from pH 4-7 (18 cm) and loaded with 100 ug of total cellular protein. For the second dimension 12-14 %T SDS-PAGE gels were used. Molecular weight is indicated with MW (kDa). Proteins were visualised by silver staining. Figure 7B. 2-DE map of one day at 12ºC cold treated Doongara anther proteins at the early stage of microspore development. Cold responsive protein spots were assigned arbitrary numbers. Up regulated, down regulated and newly induced protein spots were marked with rectangles, circles and double rectangles respectively. Isoelectric focusing in the first dimension was on IPG strips with a linear gradient ranging from pH 4-7 (18 cm) and loaded with 100 ug of total cellular protein. For the second dimension 12-14 %T SDS-PAGE gels were used. Molecular weight is indicated with MW (kDa). Proteins were visualised by silver staining. Figure 8. 2-DE map of two days at 12ºC cold treated Doongara anther proteins at the early-middle stage of microspore development. Cold responsive protein spots were assigned arbitrary numbers. Up regulated, down regulated and newly induced protein spots were marked with rectangles, circles and double rectangles respectively. Isoelectric focusing in the first dimension was on IPG strips with a linear gradient ranging from pH 4-7 (18 cm) and loaded with 100 ug of total cellular protein. For the second dimension 12-14 %T SDS-PAGE gels were used. Molecular weight is indicated with MW (kDa). Proteins were visualised by silver staining. Figure 9. 2-DE map of four days at 12ºC cold treated Doongara anther proteins at the middle stage of microspore development. Cold responsive protein spots were assigned arbitrary numbers. Up regulated, down regulated and newly induced protein spots were marked with rectangles, circles and double rectangles respectively. Isoelectric focusing in the first dimension was on IPG strips with a linear gradient ranging from pH 4-7 (18 cm) and loaded with 100 ug of total cellular protein. For the second dimension 12-14 %T SDS-PAGE gels were used. Molecular weight is indicated with MW (kDa). Proteins were visualised by silver staining. Figure 10A. 2-DE map of untreated Doongara anther proteins at the early stage of microspore development. Isoelectric focusing in the first dimension was on IPG strips with a linear gradient ranging from pH 4-7 (18 cm) and loaded with 100 ug of total cellular protein. For the second dimension 12-14 %T SDS-PAGE gels were used. Molecular weight is indicated with MW (kDa). Proteins were visualised by silver staining. Figure 10B. 2-DE map of four days at 12ºC cold treated Doongara anther proteins at the middle stage of microspore development (nitrogen applied at panicle initiation stage. Cold responsive protein spots were assigned arbitrary numbers. Up regulated, down regulated and newly induced protein spots were marked with rectangles, circles and double rectangles respectively. Isoelectric focusing in the first dimension was on IPG strips with a linear gradient ranging from pH 4-7 (18 cm) and loaded with 100 ug of total cellular protein. For the second dimension 12-14 %T SDS-PAGE gels were used. Molecular weight is indicated with MW (kDa). Proteins were visualised by silver staining. Figure 11. 2-DE map of four days at 12ºC cold treated HSC-55 anther proteins at the middle stage of microspore development. Isoelectric focusing in the first dimension was on IPG strips with a linear gradient ranging from pH 4-7 (18 cm) and loaded with 100 ug of total cellular protein. For the second dimension 12-14 %T SDS-PAGE gels were used. Molecular weight is indicated with MW (kDa). Proteins were visualised by silver staining.

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3.4.2 One day cold-temperature treatments Anther samples (C sample) were harvested after one day of cold-temperature treatment initiated as the individual plants attained an AD of – 15 mm. The transition to this temperature prevented any change in AD or developmental stage over the time course, thus anthers from control plants were harvested when they attained the same AD (– 15 mm) as had the cold-treated ones after one day. These anthers were harvested at an AD that correlated to the tetrad to early stage of young microspore development. A minimum of two repeats of silver stained 2-DE maps in the pH range pH 4-7 were generated and compared with controls. Although gels using control samples were not very promising due to extraction variability caused by the small amounts of anther material that could be harvested, at least 10 protein spots were detected as consistently altered by the cold treatment (Figures 7A and 7B). These protein spots were also evident in the 4 days cold treatment of C sample material.

3.4.3 Two days of cold-temperature treatment Anther samples (B sample) were either harvested from plants at an auricle distance (AD) of around – 5 mm for the control treatment, or after two days of cold-temperature treatment initiated when individual plants had attained an AD of –15 mm. These pooled anthers were at the early to middle stage of microspore development. At least two repeats of silver stained 2-DE maps in the pH range of 4-7 were generated and compared with controls (Figure 8) 3.4.4 Four days of cold-temperature treatment Anther samples (B sample) were either harvested at an auricle distance (AD) of 0 for the control treatment, or after four days of cold-temperature treatment initiated when individual plants had attained an AD of –15 mm. These anthers were at the middle stage of microspore development. Silver stained 2-DE maps in the pH range pH 3-10, 4-7 and 6-11 were generated and compared with control (Figures 9). These 4 days treated B samples were extensively studied although they were not as tightly controlled in the synchrony of their developmental stage as the C samples. 3.4.5 Anther pellet extracted samples Pellets remaining after solubilisation in standard lysis/loading buffer from the B samples detailed above (2.2) were re-extracted with an SDS based solubilisation buffer. Data not shown. 3.4.6 Days cold-temperature treatment after nitrogen application at PI To some plants of the C samples, nitrogen was applied at the panicle initiation (PI) stage (see methodology). This was to enhance the cold-induced sterility by top dressing with N-fertiliser. Thus, we were expecting to see an enhancement of the changes due to the cold treatment. Anther samples (C sample) were either harvested at an auricle distance (AD) of around 0 for control treatment, or just after 4 days of cold treatment of the plants from an AD of – 15 mm. These anthers were at the middle stage of microspore development. At least two repeats of silver stained 2-DE maps in the pH range of pH 4-7 and 5-6 were generated and compared with controls (Figures 10A and 10B). These samples were also extensively studied and they were one of the most synchronously controlled sample sets available. 3.4.7 Four days of cold-temperature treatment and four days recovery Anther samples (B sample) were either harvested at an auricle distance (AD) of 60-110 mm for the control treatment, or four days after four days of cold-temperature treatment initiated when individual plants had attained an AD of –15 mm. These anthers were at the middle to late stage of microspore development. Coomassie stained 2-DE maps in the pH range pH 4-7 were generated and compared with control (Data not shown). This was done to reveal down-stream affects of cold treatment, which ultimately lead to sterility of the pollens in the rice anthers. Two up regulated protein spots were identified as COA O-Methyltransferase and adenosyl methionine synthetase (see figure 12).

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3.4.8 Four days of cold-temperature treatment of relatively cold tolerant Hungarian cultivar HSC-55 Anther samples (C sample) from HSC-55 were either harvested at an auricle distance (AD) of between -80 to -60 mm for the control treatment, or after four days of cold-temperature treatment initiated when individual plants had attained an AD of -100 to -90 mm. These anthers were at the middle stage of microspore development. The Hungarian Cultivar HSC-55 was reported as relatively cold tolerant rice cultivar and this was also proved in our experiments (see 3.2). Silver and coomassie stained 2-DE gels were generated on which we had run proteins in the pH range pH 4-7 from anthers of control and cold treated HSC-55 plants (Figure 11). This was done to reveal and correlate the changes displayed in proteome maps of cold sensitive cultivar Doongara. The first anther proteome maps of Doongara and HSC-55 have been established. The anther proteome maps of Doongara plants grown in controlled and cold treated conditions are shown in the figures above. Excellent reproducibility and resolution of protein spots after silver staining was obtained. In analysing the B and C samples, Melanie image analysis of typical gels of Doongara anther proteins revealed over 4,000 protein spots within a pH range of 4-7 (approximately 3,800 spots) and pH 6-11 (400 more) and the size range of 10-120 kDa. 3.5 Detection of cold responsive proteins We have performed 1 day, 2 days and 4 days of cold-treatment at 12ºC for anthers at the cold-sensitive tetrad/young microspore stage and extracted proteins from microdiscected anthers for proteome analysis. Analysis of the gels reveals that while the global expression pattern of anther proteins was largely unaltered, 59 protein spots were observed as altered by 1 day, 2 days 4 days and 4 days N@PI (nitrogen applied at panicle initiation stage) of cold-treatment at 12ºC. These were results from at least two independent repeat experiments of the Doongara B and C samples. Among them, 47 protein spots were up regulated, 9 protein spots were down regulated and only one protein was newly induced. Repeat experiments using samples harvested under a more strictly controlled environment (C sample) revealed that only about 13 proteins as altered by cold treatment 4 days N@PI (nitrogen applied at panicle initiation stage) of cold-treatment at 12ºC. Two of them were down regulated, nine of them were up regulated and a single one was newly induced. These results are summarised in Tables 2.

Table 2. Summary of Cold Responsive Proteins

Spot No. Change Mr / pI Prediction of Function

1 � 1D 87.9/6.31 2 � 1D 80.4/6.31 3 � 1D � 2D 42.4/5.47 4 � 1D 42.0/5.60 5 � 1D 37.2/4.58 6 � 1D 34.1/5.04 7 � 1D 36.7/5.26 8 � 1D � 2D 32.0/5.70 9 � 1D � 4D 30.8/5.69 10 � 1D 29.3/4.43 11 � 1D � 4D 28.3/4.41 12 � 1D 25.9/4.45 13 � 1D 26.4/4.54 14 � 1D 25.2/4.56 15 � 1D 27.7/4.75

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16 � 1D 25.7/5.28 17 � 1D � 4D@PI 25.0/5.58 cystine synthase 18 � 1D �2D � 4D 26.0/6.31 ß-6-subunit of 20S Proteasome

Table 2. Summary of Cold Responsive Proteins (continue) Spot No. Change Mr / pI Prediction of Function 19 � 1D 22.6/4.38 20 � 1D 19.6/5.39 21 � 1D � 4D 19.5/5.55 22 ⇑ 1D ⇑ 2D ⇑ 4D ⇑ 4D@PI 19.0/5.57 Translation initiation factor-5A 23 � 1D 17.8/5.29 24 � 1D 16.6/5.36 Ribosomal S12E 25 � 1D � 4D@PI 17.0/4.35 15 kDa H protein of glycine

decarboxylase complex 26 � 1D � 4D@PI 16.3/4.34 Cytochrome C oxidase subunit VB 27 � 2D � 4D@PI 42.1/5.44 Monodehydroascorbate reductase 28 � 2D � 4D@PI 37.8/4.53 Calreticulin 29 � 2D 36.1/4.76 30 � 2D 33.7/4.81 31 � 2D 33.7/4.87 32 � 2D 35.8/5.16 33 � 2D 34.8/5.66 34 � 2D 35.6/5.82 35 � 2D 38.6/6.30 36 � 2D 30.5/4.47 37 � 2D 31.2/4.70 38 � 2D 31.5/4.96 39 � 2D 32.9/5.50 40 � 2D 32.1/5.61 41 � 2D 31.5/5.64 42 � 2D 28.1/5.83 43 � 2D 26.3/6.18 44 � 2D 16.6/5.36 Ribosomal S12E 45 � 4D 80.4/5.73 46 � 4D 80.4/5.81 47 � 4D 75.8/85.82 48 � 4D 29.9/4.78 49 � 4D 29.9/4.86 50 � 4D 40.2/5.21 51 � 4D 33.2/5.80 52 � 4D 33.7/5.83 � 4D 27.6/5.30 54 � 4D 20.0/5.75 55 � 4D@PI 35.4/4.94 56 � 4D@PI 34.3/5.72 57 � 4D@PI 32.1/5.64

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58 � 4D@PI 26.7/6.29 59 � 4D@PI 13.6/4.47 T42 gene (ns-LTP) Table 2. Summary of cold responsive proteins of 1 day, 2 days 4 days and 4 days N@PI (nitrogen applied at panicle initiation stage) of cold-treatment at 12ºC in cold sensitive Doongara cultivar. Cold responsive protein spots were assigned arbitrary numbers. Up regulated, down regulated and newly induced protein spots were marked with �, � and ⇑ symbols respectively. Proteins of interest are highlighted in bold. 3.6 N-terminal microsequencing, de novo sequencing and Peptide Mass Fingerprinting Amino-terminal sequences of between 3-15 amino acid residues were obtained for 17 out of 27 protein spots blotted onto PVDF membranes (Table 3). No sequence data could be obtained for the rest of the proteins due to either the N-terminal being blocked or not enough protein being available. Sequence homology searches revealed that 12 proteins displayed significant sequence similarity to previously identified proteins or hypothetical proteins encoded by open reading frames (ORFs). In most cases, the Mr of the sequenced proteins, and in some cases the pI, were in good agreement with the theoretical mass (Mm) and pI of the matched proteins. In addition, the matching sequences occurred either at the N-terminal region or just after a cleavage site for a potential signal peptide, further supporting the validity of this protein identification approach. The most significant identities (the highest degree of homology) were determined for 5 out of the 17 spots for which N-terminal sequence information was obtained.

Table 3. Identification of Rice anther proteins by N-terminal sequencing Spot N-terminal sequence Homology (Identity) Or Access

No. MW/pI (d)

Mr/pI (e)

m1-1 CRNVCRRCMYRSTIR Biotin synthase related protein (58%)(c)

Mt O27266 40.9/5.11 40.0/4.51

m1-3 AARAVKETTG No match 21.4/4.85 m1-6 AVEEITE(E/A)PR No match 18.3/5.46 m1-7a

STVLDGLKYSSSHE 15 kDa H protein of glycine decarboxylase complex (93%) (a)

Os O22535 13.6/5.87 16.6/4.53

m1-7b

SGDKPATVEDVMPIA Cytochrome C oxidase subunit VB (100%) (a)(b)

Os P92683 16.1/4.65 16.6/4.53

m2-1 QEIR no match 62.6/4.93 m2-7 TPT(L)EF no match 27.6/4.98 m2-9 AVQCGQVMQLMAPCM T42 gene (100%) (a) Os G904318 9.4/4.49 11.3/4.67 m2-10

AATTCVASLLELSPC hypothetical rice microspore protein (100%) (Osc6)(a)

Os TC20840 3.91/9.61 12.1/4.99

al-5 PEFNPYENSLNFLLA No match 18.8/6.89 m3-2 SDGIFVFGRFLPTGR No match 26.3/4.63 17 EVFFQEKFEDGWESR Calreticulin (100%) (a) Ma Q9SP22 45.6/4.48 37.9/4.53 39 SRPVXVTTQG No match 23.5/5.40 50 XPVESPAASV No match 16.7/5.36 41 SKREHKQT No match 19.3/4.63 25 SGTTTTXLTLHHHRT No match 33.5/5.69 29 MPG No match 26.4/6.18 Table 3. Identification of Rice anther proteins by N-terminal sequencing. Proteins selected for N-terminal microsequencing were assigned arbitrary numbers. For proteins which have no SWISS-PROT accession number, the EMBL/GenBank accession number or the TIGR gene index is given. (a): Sequence alignment inferred the proteolytic cleavage of a signal peptide from these proteins. (b): rice mitochondrion genome coded. (c): the percentage is in 12 amino acid only, otherwise for all the amino acids. (d): theoretical molecular mass (kDa) & pI, (e): calculated molecular weight (kDa) and pI. Ma: maize, Mt: Methanobacterium thermoautotrophicum, Or: Organism, Os: Oryza sativa.

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Complete or partial sequence information was obtained for 4 out of 6 protein spots analysed by tandem mass spectometry analysis with the PE SCIEX QSTAR Hybrid LC/MS/MS Quadrupole TOF System (Table 4). Complete protein sequence information was obtained for one of the protein spots by obtaining sequence of peptide fragments derived from partial tryptic cleavages that could be assembled with a five-fold overlap. One or two peptide full sequence tags were obtained for the other proteins. They all gave very high matches to rice proteins in the databases, thus making it possible to get their identities.

Table 4. Identification of rice anther proteins by tandem mass spectrometry analysis No peptide sequences Homology (Identity) Or Access No MW/pI (a) Mr/pI (b) 1 NGYIVIK,

DDLRLPTDDSLLGQIK Translation Initiation Factor EIF-5A (100%)

Os Q9ZSU2 17.5/5.77 18.9/5.57

2 Complete sequence (221 Aa) ß-6-subunit of 20S Proteasome (100%)

Os O64464 24.3/6.43 25.9/6.3

4 DAAAGFPAVDFSR T42 gene (LTP) (100%)

Os G904318 9.4/4.49 11.3/4.67

5 ALCAEHNVHLVTVPSAK Ribosomal protein S12E (100%)

Ba Q9XHS0 15.3/5.35 16.7/5.36

Table 4. Identification of Rice anther proteins by tandem mass spectrometry analysis. Proteins selected for tandem mass spectrometry analysis were assigned arbitrary numbers. Peptide sequences indicates full sequences of tryptic digested peptides. Complete sequence was deduced from alignment of all fragments analysed with at least five times overlapping. For proteins which have no SWISS-PROT accession number, EMBL/GenBank accession number is given (a): theoretical molecular mass (kDa) & pI, (b): calculated molecular weight (kDa) and pI. Ba: Barley, Or: Organism, Os: Oryza sativa. Of 120 proteins processed by MALDI-TOF analysis to generate peptide mass fingerprints, only 20 could be identified with high confidence (Table 5). Matches assigned to the peptide mass fingerprint were based on multiple criteria including which species the matched protein was derived from, the number of PMF matches, % sequence coverage, the number of missed cleavages, the overall score, and compatibility of isoelectric and molecular mass of the analysed and database protein. In summary, we were able to identify with high confidence the presumptive function of more than 28 proteins through database matching after N-terminal sequencing (5), tandem mass spectrometry analysis (4) and peptide mass fingerprinting (20) as shown in the figure 12 and tables 3, 4 and 5. Analysis of the gels reveals that while the global expression pattern of anther proteins was largely unaltered, 59 protein spots were observed as altered by 1 day, 2 days 4 days and 4 days N@PI (nitrogen applied at panicle initiation stage) of cold-treatment at 12ºC. These were results from at least two independent repeat experiments of the Doongara B and C samples. Among them, 47 protein spots were up regulated, 9 protein spots were down regulated and only one protein was newly induced. Repeat experiments using samples harvested under a more strictly controlled environment (C sample) revealed that only about 13 proteins as altered by cold treatment 4 days N@PI (nitrogen applied at panicle initiation stage) of cold-treatment at 12ºC. Two of them were down regulated including the t42 gene product (a non-specific lipid transfer protein) and calreticulin. Ten of them were up regulated, including the ß-6-subunit of the 20S proteasome, a 15 kDa H protein of the glycine cleavage system, cytochrome C oxidase VB subunit, cystine synthase, Ribosomal S12E and monodehydroascorbate reductase. A translation initiation factor EIF-5A was detected as a single newly induced protein spot. These results are summarised in Tables 2.

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Table 5. Identification of rice anther proteins by peptide mass fingerprinting

No. Identity Or PM SC Access No. MW & pI # Mr & pI * 1-7 HSC 70 Pe 9 23% P09189 71.2/5.11 64.7/5.23 1-8 dnaK-type molecular chaperone BiP Os 15 32% O24182 73.5/5.30 65.3/5.3 1-10 Vacuolar ATP synthase SU-A Ca 15 28% P09469 68.8/5.29 58.6/5.35 1-12 Glucose-6-P Isomerase Sp 5 12% O82058 61.4/5.21 53.6/4.93 2-2 Monodehydroascorbate reductase Os 15 44 % BAA77282 43.0/5.36 42.3 / 5.44 2-3 Actin Ps 12 45 % U76190 41.7/5.31 40.8 / 5.46 2-10 vac-ATP synthase catalytic su A Ma 16 28% p49087 62.0/ 5.88 35.6 /5.46 2-10 Putative adenylate kinase Os 10 35 % 6063552 35.3/8.97 35.6 /5.46 2-14 Ascorbate peroxidase " 14 61 % P93404 27.2/5.42 28.7 / 5.69 2-15 Triosephosphate isomerase " 9 45 % P48494 27.1/5.38 29.2 / 5.62 2-16 Ascorbate peroxidase " 9 45 % P93404 27.2/5.42 29.2 / 5.53 2-18 Dehydroascorbate reductase " 5 38% 6939839 23.6/5.65 23.9 / 6.20 2-48 adenosyl methionine synthetase " 12 46% P93438 42.9/5.68 41.7 / 6.00 2-53 Caffeoyl-COA O-Methyltransferase " 12 58 % Q9XGP6 28.4/4.85 33.7 / 4.86 2-55 ATP synthase á-chain, Mt " 8 28 % P15998 55.3/5.85 51.0 / 6.17 2-56 cystine synthase " 9 33% 4574139 34.3/5.35 26.1 / 5.57 2-57 Ascorbate peroxidase " 9 45% P93404 27.2/5.42 25.4 / 5.16 2-58 Anti-freeze like protein " 5 6721520 80.8/7.61 23.2 / 5.19 2-64 Heat shock protein 70 " 8 25% Q40693 71.1/5.13 70.7 / 5.30 2-92 Translation initiation factor-5A " 4 40% Q9ZSU2 17.5/5.77 .9 / 5.57 Table 5. Doongara anther proteins identified by peptide mass fingerprinting with high confidence. Ca: Carrot, HSC: heat shock proteins, Ma: maize, Or: organism, Os: Oryza sativa, Pe: Petunia, PM: Peptides matched, Ps: Pisum sativum, SC: sequence coverage, Sp: Spinach, su: sub unit. #: theoretical molecular mass (kDa) & pI, *: calculated molecular weight (kDa) and pI. Odd molecular weight or pI indicated with bold. For proteins which have no SWISS-PROT accession number, the EMBL/GenBank accession number is given. 3.7 Use of cold responsive anther proteins for antibody production Three proteins that were changed in amount following cold treatment were selected for use in producing antibodies. The proteins selected were a) T42 gene product (ns-LTP); b) 15 kDa H-protein of glycine decarboxylase complex and Cytochrome C oxidase subunit VB and c) ribosomal protein S12E. All 4 proteins have been presented as antigens in rabbits according to the method presented earlier, however we have yet to detect an immune response from sera taken from these rabbits.

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Discussion 4.1 Explanation of the approach In previous studies, research largely by the Japanese had identified some physiological and cytological properties of cold damage at the reproductive stage of the plant development which leads to male sterility in rice, as described in introduction. This research indicated the most cold susceptible organ (which is the anther), the most sensitive stage (which is from tetrad to early microspore stage), the synchronicity of microspore development of the spikelets from the top three branches, and the effect of different temperatures on the sterility of the plant. In our experimental system, and using the Australian variety Doongara, we also find similar results. This has permitted us to focus on anthers from the top three braches of the panicle at the young microspore stage and use a 12ºC cold treatment as it gives very high sterility to the spikelets of interest. In spite of a number of studies conducted at the molecular level to find out which genes or which pathways are responsible for cold-induced sterility, the underlying mechanisms that causing cold-induced male sterility are still poorly understood (see introduction). Furthermore, it is not known if the tapetal abnormalities, the tapetal hypertrophy or the biochemical changes seen are the causes of the processes which lead to pollen sterility or their consequences. We focused on the identification of the gene products that are affected by the first few days of the process of sterility triggered by cold temperatures in rice anthers. To identify which gene products are involved in this process of cold-induced male sterility, we have used the proteome analysis technique to investigate the effect of cold-treatments on rice anther proteins. Proteomics is the study of protein properties (expression level, post-translational modification, interactions etc) on a large scale to obtain a global, integrated view of disease processes, cellular processes, cell response to the environmental stresses and cell networks at the protein level. A major advantage of 2-D protein electrophoresis is the ability to separate and visualise the entire cellular complement of proteins of a specific stage of a specific tissue and to subtract this image from untreated or control tissues to identify proteins that are differentially expressed. In recent years, general proteome maps have been constructed for numerous organisms with high success in protein identification including proteome maps for different rice tissues (Tsugita et al. 1994 and 1996; Komatsu et al., 1999). However, the use of this technique to investigate the molecular mechanisms of cold-induced sterility in rice has never been previously reported. In this project, we extensively studied the rice anther proteome and its response to cold treatment. 4.2 Rice anther proteome map established The genome of the rice plant is 430 Mb. It is estimated that there are about 24,000 genes expressed in rice (Yoshimura, 1998). Our results show that up to 20% of the genome can be displayed by 2-DE of proteins from anthers at the specific stage (young microspore stage) of the specific organ (anther) that is the critical site for cold-temperature damage. This is the first report of a complete rice anther proteome. We investigated the effect of cold-treatment upon anther proteins. We have been able to apply the most recent advances in Proteomics technology not only to resolve more proteins but also to reveal their identities. By applying techniques that amplify sub-fragments of the proteome, we are now able to display more that 4,000 proteins 4.3 Most of the anther proteins are unaltered by cold treatment Greater than 4,000 separate proteins extracted from Doongara anthers have been resolved as mentioned above and cold temperature treatments of 2 and 4 Days have been extensively studied to reveal a small subset of proteins which are cold responsive. While the global expression pattern of anther proteins was largely unaltered after cold-treatment, 59 proteins (1% of the total proteins visualised) were observed as altered after comparison of many independent experiments. Among them, one protein was newly induced, 47 protein spots were up regulated and 9 protein spots were

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Figure 12. Summary of identified cold responsive anther proteins. The image is the 2-DE map of four days at 12ºC cold treated Doongara anther proteins at the middle stage of microspore development (nitrogen applied at panicle initiation stage. Identities of landmark (unchanged) proteins also shown. Up regulated, down regulated and newly induced protein spots were marked with rectangles, circles and double rectangles respectively. Isoelectric focusing in the first dimension was on IPG strips with a linear gradient ranging from pH 4-7 (18 cm) and loaded with 100 ug of total cellular protein. For the second dimension 12-14 %T SDS-PAGE gels were used. Molecular weight is indicated with MW (kDa). Proteins were visualised by silver staining. Down regulated in anthers of Doongara cultivar. In relatively cold tolerant Hungarian cultivar HSC-55, these changes were not detected (table 2. And figure 12.) 4.4 The proposed relation between the functions of cold responsive proteins and their potential roles in microspore development and their response to cold We were able to identify more that 20 proteins including 11 cold responsive proteins. Among the landmark proteins (specific, easily detectable reference proteins not differentially displayed by the cold treatment), there were some housekeeping proteins. Their identities include actin, ascorbate peroxidase (at least three different isoforms) implying basic roles for these proteins in the maintenance of cell proliferation, regardless of cold treatment in the rice anthers. However these proteins were observed as non cold-responsive proteins. Below are the descriptions of identified cold-responsive proteins that have functions potentially relevant to the cold induced sterility. 4.4.1 T42 gene product (plant ns-LTP) This protein is down regulated by cold treatment in the cold sensitive cultivar but not in the relatively cold tolerant cultivar as described in results (3.2 and 2.3). The stamen specific rice T42 gene product is predicted to a member of the plant non-specific lipid transfer protein (ns-LTP) family. All members of this family share very similar structure. The T42 gene product has a signal peptide of 28 amino acid, molecular weight is about 9 kDa (encoded by 92 amino acids after the signal peptide cleavage) and contains 8 cystines of which the positions are strongly conserved in all plant LTP families. A proposed function of LTPs is to transfer phospholipids & galactolipids across membranes. The LTP proteins may be involved in membrane biogenesis and lipogenesis. It is reported that a plant lipid transfer gene is up regulated by low temperature in the winter cultivar of barley but not in the spring cultivar (White et al. 1994). The LTP gene family in rice constitutes a complex multigene family with at least seven members grouped into three, possibly four, differentially regulated subfamilies. Although the T42 gene product shares many similar properties with other plant ns-LTP families, there is some dissimilarity between them. While the most of the LTP proteins are basic proteins (pI above pH 9.0), the rice T42 gene product was revealed to be an acidic (pH4.6) protein. The critical amino acid Tyrosine79 at which a side-chain divides the hydrophobic cavity into two parts in normal ns-LTP, is replaced with Leucine. This could indicate some difference in the function of this protein. Also of interest is the identification in Brassica napus of putative lipid-transfer proteins (Koltunow et al. 1990; Foster et al., 1992; Crossley et al. 1995). Pollen grains contain several lipidic structures, which play a key role in their development as male gametophytes. The elaborate extracellular pollen wall, the exine, is largely formed from acyl lipid and phenylpropanoid precursors, which together form the exceptionally stable biopolymer sporopollenin. The deposition of the exine begins soon after the completion of meiosis II. It is possible that these anther-specific LTPs participate in the transport of fatty acids and/or other sporopollenin precursors from the tapetum to the microspore during the early stage of exine formation. Consequently, a reduction in the amount of this protein in response to cold would reduce the degree to which the pollen grain can lay down sporopollenin, and this may play a role in the subsequent pollen sterility. 4.4.2 ß-6-subunit of 20S Proteasome and Calreticulin Proteasomes function mainly in the ATP-dependent degradation of proteins that have been conjugated with ubiquitin. There are two types of eukaryotic proteasome, with sedimentation coefficients of 20 S and 26 S. The 26 S complex (with a molecular mass greater than 1500 kDa) consists of the 20 S complex (ca 700 kDa), which is itself composed of 13-15 different subunits, and

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a characteristic set of other protein components (35-110 kDa). The multicatalytic properties of proteasomes appear to be advantageous for the rapid breakdown of certain proteins, such as abnormal proteins and proteins that turn over rapidly (Coux et al., 1996). Calreticulin is a calcium binding protein that located in endoplasmic reticulum. Calreticulin is proposed to have several functions including a role in Ca2+ -binding and storage, Ca2+ -signalling, cell adhesion and gene expression (see reviews by Crofts et al. a & b 1998). 4.4.3 Vacuolar ATP synthase catalytic sub unit A, cystine synthase, Caffeoyl-COA O-Methyltransferase and adenosyl methionine synthetase These proteins were observed as up regulated following four days at 30C/20C after 4 days of cold treatment at 12oC. The timing suggests that these proteins are later consequences of the earlier lowered temperature, which ultimately lead to sterility of the pollen in the rice anthers. It is reported that Caffeoyl-COA O-Methyltransferase is the key element of an alternative pathway for lignin biosynthesis in zinnia cells during tracheary element formation (Maury et al., 1999, Ye et al., 1994; Ye and Varner, 1995), thus, suggesting potential role in exine formation. 4.4.4 Glycine cleavage system H-protein and Cytochrome C oxidase VB subunit These two proteins were increased by cold treatment as described in the results (section 3.4). H-protein is a subunit of the glycine cleavage system located in mitochondria that catalyses the degradation of glycine and it is consists of the L-,P-, H-, and T-proteins. The H protein plays a central role in communication among the other enzymes by shuttles the methylamine group of glycine from the P protein to the T protein. Cytochrome C oxidase VB subunit is a small subunit of Cytochrome C oxidase (COX) encoded by the mitochondrial genome and also located in mitochondria. Cytochrome C oxidase catalyses following reaction: 4 Ferrocytochrome C + O2 = 2 H2O + 4 Ferricytochrome C. Chillling temperatures (above 0ºC) have been reported to lower expression and activity of COX in the mitochondrial inner membranes of a chilling-susceptible genotype of maize (Prasad et al., 1994). However the decline in this activity was partially counterbalanced by an increase in the rate of the alternative oxidase (Prasad et al., 1994) 4.4.5 EIF-5A EIF-5A were revealed as the only cold induced protein in the cold sensitive cultivar Doongara, and was consistently expressed after 1, 2 and 4 days cold treatment at 12ºC. However, this protein did not change in expression in the relatively cold tolerant HSC-55 indicating potential involvement of this protein in the process leading to cold induced sterility in rice.. Eukaryotic translation initiation factor 5A (eIF-5A) is a ubiquitous protein found in all eukaryotic cells (Park et al., a1993). It is proposed that hypusine formation in eIF-5A may directly affect the expression of a selective set of genes involved in the G1-S boundary in the eukaryotic cell cycles. (Park et al., b1993, Mehta et al., 1994). The precise role of eIF-5A in protein biosynthesis in not known but it functions by promoting the formation of the first peptide bond. 4.4.6 Summary of the cold responsive proteins and their potential involvement in microspore development In conclusion, these cold-responsive proteins are involved in protein synthesis and folding, lipid biogenesis, protein breakdown and energy metabolism. These functions are all potentially involved in processes which if perturbed may give rise to the effects seen with cold-temperature treatment. These would affect mitochondria (and its inner membrane), endoplasmic reticulum, ribosomes, membranes, plastids and cell walls (exine formation), all of which have been observed to be affected by cold treatment. In the past, it was suggested that cold induced male sterility might be caused by disruption of sugar metabolism. However our results indicate that there are a number of additional cell functions that are being varied by cold. Some other pathways might be affected by low temperature treatment. The identities obtained for the matched proteins suggest that these pathways are involved in protein

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synthesis and folding, lipid biogenesis, protein breakdown and energy metabolism as described above. 4.5 Antibody production and Immunohistochemistry 4.5.1 Antibody production Production of antibodies against specific cold-responsive anther proteins allows identification of the anther tissues that contain the cold-responsive proteins and characterisation of the protein and its interactions with other proteins. Antibody probing of selected cold responsive proteins by western blotting and immunohistochemistry detection may reveal tissue localisation of the protein and its precise expression upon cold treatment. This may reveal the role of these proteins in the cold-induced male sterility in rice. Three rice anther proteins were selected for antibody production and small but usually sufficient amounts of the antigen were generated. They were injected into rabbits in an attempt to obtain highly specific polyclonal antibodies. The preliminary results showed no immune response to these rice anther proteins by several western-blot detection with ECL (horseradish peroxidase conjugated secondary antibody with enhanced chemiluminescence detection kit). This work will continue past 30 June 2000 with funding from the CRC for Sustainable Rice Production. Other selected cold-responsive anther proteins are in the process of being bulked up to generate sufficient material to produce an immune response. However, the small amount of other cold-responsive anther proteins available may limit antibody production as described above. To guard against this possibility, internal sequence information from these proteins may be used to generate synthetic peptides to overcome the limitation of protein amount in the antibody production. The protein 51 (ns-LTP) gave 100% homology in the first 15 amino acid and has a very similar MW and pI value to the product translated from the rice anther specific gene T42 of the Japanese rice cultivar Nippon Bare. We are making use of the sequence to clone this gene into an E.coli protein expression vector. This will provide large amounts of the cold-responsive protein against which antibodies can be raised. This work will also continue with CRC funding for the next year. Upon the successful completion of antibody production, western blotting and immunohistochemistry will be carried out. Western blotting will help to identify quantitative changes in the expression of these proteins at different stages of microspore development during control treatments and cold treatment conditions. The use of the cold sensitive cultivar Doongara and the more cold tolerant cultivar HSC55 will be continued as we wish to correlate which proteins, respond to cold temperature in Doongara, are lessened or absent in HSC-55. These comparisons will also continue with CRC funding for the next year. Production of antibodies against specific cold-responsive anther proteins allows identification of the anther tissues that contain the cold-responsive proteins and characterisation of the protein and its interactions with other proteins. 4.5.2 Immunohistochemistry Following identification of cold responsive proteins, and with the preparation of highly specific polyclonal antibodies to them in progress, we have managed to obtain a polyclonal antibody against maize LTP from Prof. Jean-Claude Kader in Université Pierre et Marie Curie, France. The use of all of these antibodies will enable a precise visual identification of the cell, cell organs and tissues types in the anther that are responding to the cold treatment. This will assist in determining the extent and timing of processes we have postulated are potentially involved in the cold-sterile phenomena..

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Implications One of the major implications of this research is the validation of the Proteomic approach. Its capacity to handle very small amounts of tissue and yet deliver identification of a few elements which are varying in a complex population of proteins is most valuable. At a more practical level, the identification of a small set of specific proteins which are cold-treatment responsive in a sensitive strain, and yet are not (or less) cold-responsive in a more cold-tolerant cultivar, indicates that these components should prove useful in giving rice breeders specific molecular outcomes to test for. The use of these proteins to raise antibodies which can be used to quantitate the amounts present at crucial, sensitive time points during development is also likely to provide new tools for marker assisted breeding.

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Recommendations There are some important findings that have been made in the undertaking of this project. Already, through the rice CRC, the results here have proved useful to the group of researchers working on a range of aspects of cold-sensitivity. The continuation of this research through the CRC will permit far more results to be obtained at a time when the CRC collaborators are making some real headway and are positioned well to benefit from a continued interaction with this project. As the CRC is now taking on much of the cold-sensitivity research that was previously funded by the RIRDC, we recommend that the RIRDC use its influence as a CRC partner to encourage the CRC to continue its multi-disciplinary approach to this phenomena and maintain regular fora for the researchers to exchange results and ideas.

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