长日光周期对水稻幼穗发育的影响及其对光敏核不育水稻育性转换的作用研究

光敏核不育水稻晚粳农垦58S具有长日照下不育、短日照下可育的特点，是目前二系法杂交水稻应用的基础。对于其长日光周期引起雄性败育的特性已得到很多实验的支持，但这种光周期反应特性是光敏不育材料所特有，还是在水稻穗发育中普遍存在，目前尚不清楚。对这一问题的认识涉及到对光敏不育性本质的了解及对这一性状的有效利用，本文对此进行了系统的研究分析。 本研究以24种水稻品种包括光敏核不育系及常规水稻品种为材料，在控制光周期下进行。即利用16h长日照处理(LD)和l0h短日照处理(SD)及其不同组合，以抽穗期、叶龄、抽穗叶片总数、花粉育性、结实率、穗长、穗粒密度为指标，结合光敏不育系幼穗发育的形态解剖学特征，探讨了在整个水稻发育中包括叶片生长、幼穗分化以及穗发育等过程中，不同材料的光周期反应特征，尤其是二次枝梗期后的穗发育过程中的光周期反应特征。此外还分析了温度与光周期反应的关系及温度在光敏不育现象中的作用，并研究了代谢抑制剂对光敏不育特征的影响。 研究表明，光周期对水稻的出叶速度基本没有影响，但对水稻的抽穗叶龄有影响，长日照使抽穗叶龄增加而延迟其穗分化及抽穗。光周期还对幼穗分化后的穗发育过程有抑制延迟，作用，影响大小因品种而异，以对早稻、籼稻的影响最弱，对晚稻、粳稻的影响最强，与其穗分化中的感光性有明显的相关性。 除对抽穗期有影响外，穗发育阶段的长日光周期还影响着穗发育的其它性状，如使穗长增加，芒较长、稳粒密度降低，花粉育性降低，结实率下降。此外植株发育的其它性状也可受到影响，如剑叶发育不良表现为叶片缺少仅有叶鞘、倒二叶生长旺盛、植株较高等。同时几组不同组合的光周期处理结果均表明，长日光周期对水稻穗发育的影响主要发生在穗发育的前5-10天即颖花原基分化期、雌雄蕊原基分化期至花粉母细胞形成期。这些结果表明水稻的光周期反应不仅表现在茎端从营养生长向生殖生长的转换上（幼穗分化），而且还表现在幼穗分化完成后的穗发育过程中。长日光周期对晚稻穗发育均有抑制效应，且日长对稳发育的影响时期与光敏核不育水稻的‘育性转换敏感期’完全一致。因此在农垦58S中引起‘光敏不育’的原因很可能不是一种特殊的光周期反应，而是该材料雄性器官发生过程不能对长日光周期做出适当的反应。 对24种不同品种水稻的光周期反应表明，不同材料光周期反应特性不同。光敏不育系农垦58S与农垦58在对长日照的反应上也有较大不同，表现为前者在短日照下穗分化较快，在自然日照下抽穗较早。这表明除了育性不同外，农垦58S与农垦58在光周期反应特征上也有所不同，然而我们认为这种不同不是农垦58S表现光敏不育的主要原因。因为本研究中还发现，光敏不育系农垦58S与其可育回复突变体农垦58S(r)在抽穗期等光周期反应特征上相当一致，但在育性反应上却有较大不同，长日照下农垦58S(r)表现为雄性可育，而农垦58S表现为雄性败育。根据上述几方面的比较，我们认为光敏不育的机制很可能在于农垦58S突变体其雄性器官发育对环境不利信号的反应能力的变弱所致。 在本研究中发现，温度对水稻穗发育的影响表现在两个方面：一方面是通过影响光周期反应强弱而起作用，如高温可加强短日照下的穗分化和发育过程，高温亦可加强长日照对穗分化发育的抑制作用;另一方面是直接对器官发生过程产生影响，如在对短日照下光敏不育系和常规稻不同温度条件下处理时的结实率比较分析发现，常规稻的结实率与其抽穗扬花期的平均温度显著负相关，而光敏核不育水稻的结实率虽与抽穗扬花期的温度有一定相关性，但更与穗发育期的平均温度呈显著负相关，二者在受温度影响的作用时期上有显著差异，因此温度也可直接对雄性器官发育起作用。区分温度对光敏不育的两方面影响，同时考虑到光敏不育机制更有可能在于光敏不育系农垦58S雄性器官发育对环境信号反应能力的变弱的假设。我们就可以较好地理解农垦58S‘光敏不育’性状经杂交转育到对光周期弱感的籼稻中所出现的‘温敏不育性’。 核酸代谢抑制剂5-FU，2-TU对SD下的幼穗分化有较强抑制作用，使幼穗分化被迟滞，而2-BrDU和蛋白质合成抑制剂CHX、CL对其影响较小。抑制剂处理也不能诱导LD下的穗分化。 短日照下，5-FU可对穗发育有强烈抑制作用，可使常规品种农垦58及光敏不育系农垦58S穗畸形，颖花减少并发育不良，穗长缩短，枝梗减少，花粉败育甚至无花粉，结实率显著降低，其有效作用时期为穗发育的二次枝梗分化期至雌雄蕊原基分化期，与长日照诱导农垦58S败育的作用时期也完全吻合，5-FU对SD下穗发育的影响还可被核酸抑制剂的恢复剂乳清酸所部分恢复。其它代谢抑制剂如2-TU、CHX、CL等也可使农垦58S育性明显降饭，而所有这些抑制剂对常规可育的农垦58及农垦58S(r)的育性影响较小，表明它们与光敏不育系对抑制信号的反应能力有显著不同。 长日照下5-FU对LD下的农垦58S的幼穗发育也有很强的抑制作用，使稳长缩短，颖数减少，但它还可使部分LD下处理植株抽穗期较LD对照明显提前，并可使农垦58S育性部分恢复而有结实，说明5-FU还可对LD的抑制作用有抑制，通过对LD抑制作用的抑制使LD下的育性转换有部分恢复。其它代谢抑制剂在穗发育前期处理LD下农垦58S叶片均可看到植株在抽穗期较LD下提前5—8天的同时，其花粉育性有不同程度的提高，在高温长日下甚至有一定程度的结实率，表明各种抑制剂均可对穗发育中的光周期作用产生影响。 总之，本研究结果表明，短日植物水稻的光周期反应不仅存在于幼穗分化上，还存在于幼穗发育和花器官发生等发育过程中。幼穗发育的光周期效应表现为抽穗期、穗长、穗粒密度、结实率等多方面的变化，作用时期以穗发育早期的花器官发生阶段影响最大。作用强弱因品种不同而异，以粳稻和晚稻中作用较强。光敏不育突变的更主要变化可能在于农垦58S的雄性器官分化发育时对环境不利信号的反应能力变弱，导致其正常发育受阻，育性不能正常表达。温度在水稻穗发育上既可通过影响光周期反应而起作用，还可直接对穗器官发育产生影响而对育性表达起作用。此外我们还发现农垦58S与农垦58不仅在雄性育性上有显著不同，而且其光周期反应特性也有较大的差异。抑制剂处理结果也支持光敏不育系农垦58S的雄性器官发生过程较农垦58更易受抑制剂影响而育性降低，而抑制剂对长日光周期抑制作用的部分解除，可以使其育性有一定程度的恢复，也表明光周期对雄性育性的影响最为显著。这些结果可以帮助我们更加全面地认识光敏不育水稻的基本特性，从而为进一步开展光敏不育的转育及应用研究提供可靠的科学依据。

晚粳水稻生长点在光周期反应中的地位

光敏核不育水稻晚粳农垦58S具有长日下不育，短日照下可育的特点。为了确定突变体NK58S突变基因的作用器官及功能。我们设计了一系列光周期处理实验，并对不同光周期处理的生长点或幼穗进行细胞学及细胞化学观察，同时选择光调节基因对其在NK58S上表达特性进行分析。材料选用突变体NK58S，及其野生型NK58S，和它的回复突变体NK58Sr，加两个籼稻品种W6154S及珍汕97，共设计十三个不同的光周期处理。根据试验分析我们发现： 第一，温度对结实率的影响，NK58S，NK58及NK58Sr表现一致，没有发现对NK58S有特异作用的温度效应。三个粳稻品种均因幼穗分化前的长日处理延迟抽穗，而使各处理粳稻品种处于不同环境条件下，引起结实率的变化。 第二，温度对花粉育性的影响较对结实率的影响小。因而用花粉育性进行不育材料的鉴定和比较较可靠。 第三，光周期处理引起生长点原套及原体组织的一定的细胞学变化，但三个粳稻品种间没有差异。生长点周围及其下节部的淀粉积累的变化，三个粳稻品种一致，没有发现不育与可育材料之间的差异。一直处于长日处理条件下的三个粳稻材料，表现出NK58S突变体生长点周围及节部淀粉积累少于NK58，和NK58Sr。 第四，就总RNA而言，三个粳稻品种在光周期处理下各样品绝对量不同，但不同光周期处理，三个粳稻品种反应一致。不同发育时期叶片内光调节基因表达丰度与总RNA水平不一致，不同基因表现出因不同发育阶段而不同的转录特点。在所选三个光调节基因的Northern印迹分析结果没有发现三个晚粳稻品种之间的差异。 第五，幼穗分化开始后的光周期反应不是农垦58S的花粉育性所特有，对NK58，及NK58Sr也有作用。光周期处理还会影响幼穗其它方面的发育。短日处理下农垦58S的育性恢复也只有农垦58的一半。 总之，我们的试验结果使我们得出光周期作用产生的信息在植物不同发育阶段一致。不同发育时期的生长点对来自该信息的作用产生不同的反应。光敏核不育的突变表型体现在生长点的变化上。突变基因的功能是感受来自不同环境因素所产生的胁迫作用。

Temporal sensitivity of rice seed development from spikelet fertility to viable mature seed to extreme-temperature

Extreme temperature during reproductive development affects rice (Oryza sativa L.) yield and seed quality. A controlled-environment reciprocal-transfer experiment was designed where plants from two japonica cultivars were grown at 28/24 ⁰C and moved to 18/14 ⁰C and vice versa, or from 28/24 to 38/34 ⁰C and vice versa, for 7-d periods to determine the respective temporal pattern of sensitivity of spikelet fertility, yield, and seed viability to each temperature extreme. Spikelet fertility and seed yield per panicle were severely reduced by extreme temperature in the 14 d period prior to anthesis; and both cultivars were affected at 38/34 ⁰C while only cv. Gleva was affected at 18/14 ºC. The damage was greater the earlier the panicles were stressed within this period. Later-exserted panicles compensated only partly for yield loss. Seed viability was significantly reduced by 7-d exposure to 38/34 ⁰C or 18/14 ⁰C at 1 to 7 and 1 to 14 d after anthesis, respectively, in cv. Gleva. Cultivar Taipei 309 was not affected by 7 d exposure at 18/14 ⁰C; and no consistent temporal pattern of sensitivity was evident at 38/34 ⁰C. Hence, brief exposure to low or high temperature was most damaging to spikelet fertility and yield 14 to 7 d before anthesis, coinciding with microsporogenesis; and it was almost as damaging around anthesis. Seed viability was most vulnerable to low or high temperature in the 7 or 14 d after anthesis, when histodifferentiation occurs.

Down-regulation of RFL, the FLO/LFY homolog of rice, accompanied with panicle branch initiation

FLORICAULA (FLO) of Antirrhinum and LEAFY (FLY) of Arabidopsis regulate the formation of floral meristems. To examine whether same mechanisms control floral development in distantly related species such as grasses, we isolated RFL, FLO-LFY homolog of rice, and examined its expression and function. Northern analysis showed that RFL is expressed predominantly in very young panicle but not in mature florets, mature leaves, or roots. In situ hybridization revealed that RFL RNA was expressed in epidermal cells in young leaves at vegetative growth stage. After the transition to reproductive stage, RFL RNA was detected in all layers of very young panicle including the apical meristem, but absent in the incipient primary branches. As development of branches proceeds, RFL RNA accumulation localized in the developing branches except for the apical meristems of the branches and secondary branch primordia. Expression pattern of RFL raised a possibility that, unlike FLO and LFY, RFL might be involved in panicle branching. Transgenic Arabidopsis plants constitutively expressing RFL from the cauliflower mosaic virus 35S promoter were produced to test whether 35S-RFL would cause similar phenotype as observed in 35S-LFY plants. In 35S-RFL plants, transformation of inflorescence meristem to floral meristem was rarely observed. Instead, development of cotyledons, rosette leaves, petals, and stamens was severely affected, demonstrating that RFL function is distinct from that of LFY. Our results suggest that mechanisms controlling floral development in rice might be diverged from that of Arabidopsis and Antirrhinum.

Genotypic variation for cold tolerance during reproductive development in rice: Screening with cold air and cold water

Rice (Oryza sativa L.) plants are susceptible to low temperature during the young microspore stage, which occurs 10-12 days before heading. Low temperature at this time increases spikelet sterility which can cause massive yield loss. Increasing the cold tolerance of cultivars can reduce yield variability in temperate rice-growing environments. Two experiments were conducted in cold air screenings and two were conducted in cold water screenings to examine genotypic variation for cold tolerance, explore flowering traits related to spikelet sterility, and investigate whether the results reflect the level of cold tolerance determined previously in the field. Cold air screenings imposed day/night temperatures of 27 degrees C/13 degrees C, 25 degrees C/15 degrees C and 32 degrees C/25 degrees C following particle initiation until 50% heading, while cold water screenings maintained a relatively constant 19 degrees C. The variation in the commencement of low air temperature treatment did not have an effect on the level of spikelet sterility, indicating that exposure to low temperature during the young microspore stage was more important than the duration of exposure. Spikelet sterility of common cultivars showed a significant correlation between cold air and cold water screenings (r(2) = 0.63, p < 0.01), cold air and field screenings (r(2) = 0.52, p < 0.01) and cold water and field screenings (r(2) = 0.53, p < 0.01), indicating that cold air and cold water can be used for screening genotypes for low temperature tolerance. HSC55, M 103 and Jyoudeki were identified as cold tolerant and Doongara, Sasanishiki and Nipponbare as susceptible cultivars. There was a significant negative relationship between spikelet sterility and both the number of engorged pollen grains per anther and anther area only after imposing cold air and cold water treatment hence, it was concluded that these flowering traits were facultative in nature. In addition, cultivars originating from Australia and California were inefficient at producing filled grain with similar sized anthers containing a similar number of engorged pollen grains as cultivars from other origins. One suggested reason for this poor conversion to filled grain of cultivars from Australia and California may be associated with their small stigma area, particularly when exposed to low temperature conditions. (c) 2006 Elsevier B.V. All rights reserved.

Minimising cold damage during reproductive development among temperate rice genotypes. I. Avoiding low temperature with the use of appropriate sowing time and photoperiod-sensitive varieties

Multiple-sown field trials in 4 consecutive years in the Riverina region of south-eastern Australia provided 24 different combinations of temperature and day length, which enabled the development of crop phenology models. A crop model was developed for 7 cultivars from diverse origins to identify if photoperiod sensitivity is involved in determining phenological development, and if that is advantageous in avoiding low-temperature damage. Cultivars that were mildly photoperiod-sensitive were identified from sowing to flowering and from panicle initiation to flowering. The crop models were run for 47 years of temperature data to quantify the risk of encountering low temperature during the critical young microspore stage for 5 different sowing dates. Cultivars that were mildly photoperiod-sensitive, such as Amaroo, had a reduced likelihood of encountering low temperature for a wider range of sowing dates compared with photoperiod-insensitive cultivars. The benefits of increased photoperiod sensitivity include greater sowing flexibility and reduced water use as growth duration is shortened when sowing is delayed. Determining the optimal sowing date also requires other considerations, e. g. the risk of cold damage at other sensitive stages such as flowering and the response of yield to a delay in flowering under non-limiting conditions. It was concluded that appropriate sowing time and the use of photoperiod-sensitive cultivars can be advantageous in the Riverina region in avoiding low temperature damage during reproductive development.