Identifying genes responsible for failure of grain formation in rice and wheat under drought (G2005.02) The effect of drought stress at reproductive stage is most damaging to rice and wheat yields. Spikelet fertility has been reported to be the most drought-sensitive yield component. Among the processes involved in flowering delay and sterility, peduncle elongation, pollen sterility and anther dehiscence have been found to be important bottlenecks under drought stress. The project aimed to use rice and wheat genotypes with contrasting behaviour under stress to identify the candidate genes that underlie differences in drought tolerance. Candidate genes would be validated genetically, and allele mining would be used to identify novel alleles contributing to improved physiological traits under stress. A key hypothesis under test was that yield-forming processes and stress-adaptive responses are in conflict (as illustrated by GA/ABA antagonism) under drought stress. John Bennett, Ken McNally, Hong He, Reena Sellamuthu & Rachid Serraj IRRI DAPO Box 7777 Manila, Philippines. Rudy Dolferus. CSIRO Plant Industry, Canberra, Australia. Shoshi Kikuchi & Kouji Satoh: NIAS, Tsukuba, Japan. R. Chandra Babu Tamil Nadu Agricultural University, Coimbatore, India. Zhengqiang Ma, Nanjing Agricultural University, China. Rice reproductive-stage processes under drought Two genotypes, IR64 and Apo were used in a dry-down experiment, using the fraction of transpirable soil water (FTSW) as co-variable, to apply drought stress (DS) with similar duration and intensity at 5 different stages viz., panicle initiation (PI), spikelet differentiation (SD), pollen meiosis (PM), flowering (FL) and grain filling (GF). The response curves to FTSW at different stages showed little or no change in normalized transpiration ratio (NTR) until FTSW decrease to a value less than 0.4 (Fig. 1a). The yield reduction under drought stress differed significantly between the different reproductive stages, FL being the most sensitive stage to DS, with a decrease in relative yield around 58% and 64.5% in Apo and IR64, respectively (Fig. 1b). The yield reduction was mainly caused by the reduction of panicle fertility. The observation of the stages of spikelet sterility showed that in most cases the exserted anther but not fertilized stage was the most important process of spikelet sterility (Fig. 3). The analysis of ABA concentration in the peduncles showed the existence of a significant difference at FTSW = 0.1 for Apo and FTSW= 0.3 and 0.5 for IR64 between control and stress. When FTSW was the same, there was no difference in ABA concentration between the two cultivars (Fig. 5). Conclusions: Large genetic variation has been observed for traits associated with responses to reproductive-stage drought stress, including spikelet fertility, pollen fertility and peduncle elongation, and yield under stress. QTL mapping is being used to further dissect these traits and their association of performance under drought. Next steps and challenges: the key questions are (i) how drought- stressed rice and wheat mobilize stored carbohydrates (starch and fructans, respectively) and allocate them to protection vs growth, (ii) how the synthesis, function and degradation of ABA impact on drought tolerance, and (iii) how to identify the crucial members of multigene families, such as those encoding transcription factors, given that drought stress operates at many levels in addition to transcript abundance. Acknowledgments: We thank L Holongbayan, A Reyes, N Turigan, N Sadiasa, R Mabesa, A Los Anes, C Oca, K Ng, and N Driz for their technical support. This work was supported by a grant from the Generation Challenge Program (GCP) Fig. 2 The effects of 14-day drought stress on yield and traits of two contrasting cultivars at the different reroductive: a) relationship between NTR and FTSW; b) relative yield; c) spikelet fertility; d) grain number per panicle; e) relationship between spikelet fertility and panicle exsertion; f) relationship between PER and FTSW (n=5) ( IR64 Apo) FTSW 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 0.2 0.4 0.6 0.8 1 GF FL PM SD PI a y = 4.54x + 75.4 r = 0.74*** 0 20 40 60 80 100 -15.0 -10.0 -5.0 0.0 5.0 e y = 97.28x + 11.88 r = 0.84*** 0 20 40 60 80 100 0.0 0.2 0.4 0.6 0.8 f Fraction of transpirable soil water (FTSW) Relative yield(%) Spikelet Fertility (%) Grain No per panicle Spikelet fertility (%) Fraction of transpirable soil water (FTSW) Panicle exsertion (cm) 25 50 75 100 GF FL PM SD PI b 25 50 75 100 GF FL PM SD PI c 25 50 75 100 125 150 175 GF FL PM SD PI d Normalized transpiration rate (NTR) Peduncle elongation rate (PER) (mm d - 1 ) IR64 * * 0 2000 4000 6000 8000 0.5 0.3 0.1 rewater drought well-water Apo * 0 2000 4000 6000 8000 0.5 0.3 0.1 rewater drought w ell-w ater Peduncle ABA content (ng/g DW) FTSW Fig.5. Response of ABA content in peduncle at different drought stress level (n=3) Microarray analysis of transcription factor expression and ABA-GA antagonism in rice To elucidate the responses to drought stress in rice, a 60 oligomer microarray covering 22K unique genes based on the sequence of full-length cDNA clones was used to profile gene expression changes in shoots at the seedling stage and in peduncle at heading stage. Among 503 differentially expressed transcription factor-encoding genes, all the paralogous members of PHD and SNF2 families were up-regulated and those of Jumonji and TCP families were down-regulated by four drought stress treatments. AP2-EREBP, AUX/IAA, bZIP, C2C2-GATA, C3H, CPP, HB, HMG, HSF, MYB-related, NAC, SBP, SNF2 and Trihelix families were commonly up-regulated and Alfin-like, AUX/IAA, BES1, bHLH, bZIP, MYB, NAC, WRKY and ZIM were commonly down-regulated by drought stress. The metabolic pathway data in Rice Cyc showed that genes encoding many enzymes of sugar metabolism were down-regulated, along with genes encoding enzymes of cell-wall biosynthesis, while genes encoding enzymes of some amino acid biosynthetic pathways were up-regulated. Drought-induced ABA was involved in antagonizing GA-dependent events underlying peduncle elongation, but the biosynthetic genes related to these hormones were not clearly affected by the drought stress treatment. Allele mining The goal of this work was to identify superior alleles of key genes for drought tolerance. Nominated genes such as OsVP1, OsABF1, and the cytP450 for ABA 8’hydroxylase were amplified from a panel of 1536 diverse accessions of Oryza sativa by the polymerase chain reaction (PCR) and the amplicons were screened by EcoTILLING to detect variant forms. The haplotypes of the variants were then compared statistically with phenotyping data obtained on reproductive-stage drought tolerance on over 1000 accessions. ABA metabolism genes in drought-tolerant and –susceptible wheat cultivars We compared two Australian wheat cultivars at the reproductive stage: drought- sensitive Sundor, and drought-tolerant Sunstar. There was a dramatic decrease in grain number due to spikelet sterility when drought stress conditions coincided with the period around pollen meiosis. Spikelet fertility was close to unstressed levels when florets were stressed after meiosis. When stressed at meiosis, Sundor produced no seeds, while Sunstar still produced up to 20% of seeds. We screened wheat EST databases for genes of interest in this project: cell wall invertase, ABA biosynthetic genes (9-cis-epoxycarotenoid dioxygenase, NCED) and ABA catabolic genes (ABA-8’ hydroxylase, ABA8OH). Our search was focused on ESTs that were identified in cDNA libraries from the reproductive parts of the plant. We identified 4 wheat cell wall invertases (TAINV), 3 NCED (TANCED) and 3 ABA 8’ -hydroxylase (TAABA8OH). We confirmed using RT-PCR that these genes are expressed in wheat anthers and ovule Two mapping populations viz., CT9993 x IR62266 DH lines and Apo x Moroberekan BC1F3 lines were phenotyped under reproductive-stage drought stress in upland field conditions. Water was withheld in the stress plots for 28 days starting at panicle initiation (PI). The flowering was delayed under DS on an average of 13 days relative to control. Yield varied from 83.2 to 1481.8 kg.ha-1 under stress, and pollen fertility was positively correlated with the yield. Peduncle elongation and spikelet sterility traits were correlated with yield under drought and analyzed for QTL mapping and field validation. View publication stats View publication stats