Rapid PCD Process Promotes Early Maturity in Weedy Rice (Oryza

Background: Shorter grain-lling period and rapid endosperm development contributes to early maturity in weedy rice (Oryza sativa L. f. spontanea). However, the differences in programmed cell death (PCD) process and anti-oxidative enzymes system in the caryopsis between weedy and cultivated rice are largely unexplored. Main Text: we selected four biotypes of weedy rice and associated cultivated rice (ACR, Oryza sativa) from different latitudes to conduct a common garden experiment. The difference of PCD process between weedy rice and ACR was compared by chemical staining, and the cell viability and nuclear morphometry of endosperm cells were observed by optical microscopy, and antioxidative enzymes activity were also measured during grain lling. We found that the PCD progress in weedy rice was more rapid and earlier than that in ACR. The percentage of degraded nuclei of weedy rice were 10%-83% higher than that of ACR. Endosperm cells in weedy rice lost cell viability 2-8 days earlier than that in ACR. The anti-oxidant enzymes activity of weedy rice were lower than that of ACR during grain lling. The ability of weedy rice to scavenge reactive oxygen species is weaker than that of ACR, which may contribute to the rapid PCD process in the endosperm cells of weedy rice. Conclusion: The rapid PCD process and weaker ability to scavenge reactive oxygen species in endosperm cells lead to the shorter grain-lling period of weedy rice. antioxidative enzymes system enzyme activity between weedy rice and cultivated rice during grain lling. Our objectives to describe the rapid PCD process leading to a shorter grain-lling period in weedy rice. Our results on the differences in PCD process of endosperm cell between weedy rice and ACR may provide a new perspective for the control of weedy rice. kernel tissues cut into approximately 8-µm-thick on a rotary microtome mounted on slides coated with glycerol albumin, and then dewaxed in absolute ethanol. Drop a drop of distilled water on slide, oat the slices on the distilled water for expansion, and bake slices overnight at 30 ℃ . After slices were dewaxed overnight with 100% absolute ethanol, the they dewaxed with fresh anhydrous ethanol for 1–2 times, 2–3 h each then dried naturally for standby Dewaxed slides containing kernel tissues stained with DAPI (4’, 6-diamidino-2-phenylindole, µg/mL) Chemical MO, examined microscope Discovery nuclei


Introduction
Weedy rice (Oryza sativa L. f. spontanea) has become one of the most harmful weeds in paddy elds in the world. It is a is a plant of the genus Oryza that infests and competes with cultivated rice (Oryza sativa) in the world rice production area (Delouche et al. 2007; Azmi and Karim 2008). Weedy rice has many morphological and physiological characteristics related to weediness, such as rapid growth rate, high phenotypic plasticity, awnedness, early maturity, seed shattering, long seed dormancy and seed longevity, which facilitate seed dispersal and persistence in the paddy eld, and weedy rice has been considered one of the three worst weeds in paddy elds worldwide ( As cultivated and weedy rice share similar morphological and physiological traits, there is no selective chemical available to control weedy rice (Chauhan 2013). Our previous study found that the shorter grain-lling period promote early maturity in weedy rice compared with the associated cultivated rice. In addition, weedy rice has heavy shattering, which contribute to weedy rice escape from harvesting (Zhao et al. 2018). Furthermore, the rapid development of endosperm cells and starch grains leads to the shorter grain-lling period of weedy rice (Zhao et al. 2020). However, the relationship between the process of programmed cell death (PCD) in endosperm cells and the early maturity of weedy rice was unclear.  Fan et al. 2013). In the process of PCD of rice endosperm, the nucleus is the rst to die out, and then the cells still maintain high physiological activity Gallie 1997, 1999). In wheat endosperm, PCD occurred can occur shortly after anthesis until seed maturity Gallie 1999). DAPI (4', 6-diamidino-2phenylindole) is a kind of DNA uorescent dye with high sensitivity and speci city, which has excellent uorescence staining effect on nucleus and chromosome (Locato and De Gara 2018). Steedman's wax is a kind of low temperature wax, which can be miscible with ethanol, and can be used as para n section for serial section. Chen et al. (2012) and Wei et al. (2009) successfully used this method to observe the morphological changes of endosperm nucleus in wheat and barley during the process of PCD.
Reactive oxygen species (ROS) could destroy the normal cellular metabolism through the oxidative damage to lipids, proteins, and nucleic acids, and cause growth impairment in plants. PCD in plant cells is mainly caused by the accumulation of reactive oxygen species (Breusegem and Dat 2006). To eliminate these ROS, plants have developed a complex anti-oxidative enzymes system (AES), including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) (Nunez et al. 2003;Corpas et al. 2006;Zhang et al. 2015).
The PCD process of endosperm cells determines the length of cell cycle, and the PCD process in rice determines the length of grain lling period, and then determines the growth period of rice. There are many reports about the PCD process of cultivated rice, but the research of PCD process contribute to early maturity in weedy rice has not been reported. In previous research, we found that shorter grain-lling stages contribute to the early maturity of weedy rice, and the rapid development of endosperm cells and starch grains leads to the shorter grain-lling period of weedy rice (Zhao et al. 2018(Zhao et al. , 2020. In the present study, we used Steedman's wax embedded sections, DAPI staining, Evans blue staining and TTC staining to compare and analyze the difference of PCD process in endosperm cells between weedy rice and cultivated rice, and compare the difference of antioxidative enzymes system enzyme activity between weedy rice and cultivated rice during grain lling. Our objectives were to describe the rapid PCD process leading to a shorter grain-lling period in weedy rice. Our results on the differences in PCD process of endosperm cell between weedy rice and ACR may provide a new perspective for the control of weedy rice.

Experiment location and cultivation methods
Field trials were established at Jiangpu Experimental Farm (118°37'E, 32°02'N), Nanjing Agricultural University, China, in the summer cropping seasons (between May and November) of 2015. The eld sites have clay-loam soil with medium fertility (organic matter 2.8%, N 97 mg kg − 1 , available P 52 mg kg − 1 , available K 161 mg kg − 1 ) and pH 7.1. Echinochloa crus-galli, Leptochloa chinensis, Monochoria vaginalis and Cyperus difformis were the dominant weeds in the eld. The eld that was left fallow before the summer season was subjected to rotary tilling. According to our previous studies on the genetic diversity and morphological characteristics of various weedy rice accessions (Dai et al. 2014;Zhao et al. 2018, 2020), we selected four weedy rice accessions of different geographic origins along with their associated cultivars at the collection site. The characteristics of the four weedy rice and their associated cultivars biotypes are listed in Table 1. The materials were previously described by Zhao   (Aebi 1984). POD activity was assayed by the method described by Cakmak and Marschner (1992). SOD activity was determined through measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT), according to the method of Giannopolitis and Ries (1977).

Data analysis
The statistical analyses consisted of ANOVAs. Means were compared by the least signi cant difference (LSD) test at the 0.05 probability level. All statistical analyses were conducted using the SPSS software package (18.0; SPSS Inc., Chicago, IL, USA), and graphs were generated using Origin 8.0 (OriginLab, Hampton, MA, USA).

Comparison of nuclear morphological changes in endosperm between weedy rice and cultivated rice
The process of PCD is often accompanied by degeneration of cell nuclei. The DAPI is a highly sensitive and speci c DNA uorescent dye, which has excellent uorescence staining effect on the cell nucleus and chromosome. The endosperm cell nuclei of weedy rice and cultivated rice were in the coenocyte stage or cellularization stage, and the endosperm nucleus was small and regular spherical at 3 days post anthesis (DPA). Starch accumulated continuously in endosperm cells, and the nucleus of starch endosperm was extruded, gradually deformed and disintegrated at 5 to 9 DPA (Fig. 1).
Morphological and statistical results of starch endosperm cell nuclei of weedy rice and cultivated rice (normal nuclei, deformed nuclei and degraded nuclei) at 3, 5, 7 and 9 DPA were shown in Fig. 1 and Fig. 2. After DAPI staining, the nuclei of endosperm of weedy rice and cultivated rice in Taizhou were 100% normal nuclei at 3 DPA. The percentage of normal nuclei of TZWR was 3% lower than that of TZCR, and the percentage of deformed nuclei and degraded nuclei of TZWR were 2% and 1% higher than that of TZCR at 5 DPA, respectively. The percentage of normal nuclei and deformed nuclei of TZWR was 25%-48% lower than that of TZCR, and the percentage of degraded nuclei of TZWR were 74% and 63% higher than that of TZCR at 7 and 9 DPA, respectively (Figs. 1A1-A4, Figs. 1B1-B4; Fig. 2A).
After DAPI staining, the percentage of normal nuclei of YZWR was 44% and 15% lower than that of YZCR at 3 and 5 DPA, respectively, and the percentage of deformed nuclei and degraded nuclei of YZWR were 2%-34% higher than that of YZCR at 3 and 5 DPA. There were no normal nuclei in the endosperm cells of YZWR and YZCR, and the percentage of deformed nuclei of YZWR was 83% and 3% lower than that of YZCR at 7 and 9 DPA, respectively. The percentage of degraded nuclei of YZWR were 83% and 3% higher than that of YZCR at 7 and 9 DPA, respectively (Figs. 1C1-C4, Figs. 1D1-D4; Fig. 2B).
The endosperm cells of MMWR and MMCR were normal nuclei at 3 DPA. From 5 DPA to 9 DPA, the percentage of normal nuclei and deformed nuclei of MMWR were 5%-49% lower than that of MMCR, and the percentage of degraded nuclei of MMWR was 14%-59% higher than that of MMCR (Figs. 1E1-E4, Figs. 1F1-F4; Fig. 2C). The percentage of deformed nuclei and degraded nuclei were 18% and 2% at 3 DPA in DDWR, respectively. However, the percentage of normal nuclei was 100% in DDCR at 3 DPA. From 5 DPA to 9 DPA, the percentage of normal nuclei and deformed nuclei of DDWR were 1%-47% lower than that of DDCR, and the percentage of degraded nuclei of DDWR were10-70% higher than that of DDCR (Figs. 1G1-G4, Figs. 1H1-H4; Fig. 2D). Generally speaking, the PCD process of endosperm cell nuclei of weedy rice was faster than that of associated cultivated rice (Figs. 1, 2).

Comparison of endosperm cell viability between weedy rice and cultivated rice
Viability staining provides a means to follow the pattern and progression of cell death during endosperm development. Evans blue dye only stains cells which are no longer capable of excluding the dye, indicating a loss of membrane integrity and consequently viability. The embryos of weedy rice and cultivated rice could not be dyed blue by Evans blue, which means that the embryos were always active during endosperm development. The endosperm of weedy rice and cultivated rice was gradually dyed blue by Evans blue with the development of endosperm, that is, endosperm cells gradually lost membrane permeability and became dead cells (Fig. 3). All starch endosperm cells of TZWR were completely stained dark blue by Evans blue at 13 DPA, while those of TZCR were at 21 DPA. The starch endosperm cells of YZWR and MMWR were completely stained dark blue by Evans blue at 13 DPA, and the starch endosperm cells of YZCR and MMCR were completely stained dark blue by Evans blue at 15 DPA.
Endosperm cells of DDWR were completely stained dark blue by Evans blue 4 days earlier than that of DDCR (Fig. 3). In all, the whole starch endosperm of weedy rice was completely dyed dark blue by Evans blue 2-8 days earlier than that of associated cultivated rice, that is, endosperm cells of weedy rice lost membrane permeability and became dead cells 2-8 days earlier than associated cultivated rice (Fig. 3).
The embryo of weedy rice and cultivated rice can be dyed red by TTC, which means that the embryo has strong viability during endosperm development. The endosperm of weedy rice and cultivated rice could not be dyed red by TTC with the development of endosperm, which indicated that endosperm cells gradually lost viability (Fig. 4). The endosperm cells of DDWR could not be dyed red at 9 DPA by TTC, while the endosperm cells of weedy rice in other three places could not be dyed red by TTC at 15 DPA.
However, the endosperm cells of four cultivated rice varieties could not be dyed red by TTC at 18 DPA (Fig. 4).

Comparison of anti-oxidative enzymes system between weedy rice and cultivated rice
The anti-oxidative enzymes activity decreased gradually both in weedy rice and cultivated rice, and the CAT activity of weedy rice was signi cantly lower than that of associated cultivated rice (Fig. 5). The CAT activity levels of TZWR was 10.39-82.95 U/g lower than that of TZCR at 3-25 DPA, while similar at 30 DPA (Fig. 5A). The CAT activity of YZWR was 37.72-53.81 U/g lower than that of YZCR at 3-15 DPA, but there was no signi cant difference between YZWR and YZCR at 20-30 DAP (Fig. 5B). The CAT activity of MMWR was signi cantly lower than that of MMCR at 3-15 DPA, but there was no signi cant difference between MMWR and MMCR at 20-30 DPA (Fig. 5C). At 3 and 5 DPA, there was no signi cant difference between the CAT activity of DDWR and DDCR, and the CAT activity of DDWR was 23.62-42.20 U/g lower than that of DDCR at 10-30 DPA.
The change trend of SOD activity of weedy rice was similar to that of associated cultivated rice, and there was no signi cant difference between MMWR and MMCR. The decline rate of SOD activity of weedy rice in the other three areas was faster than that of associated cultivated rice (Fig. 6). The SOD activity of TZWR was the highest at 3 DPA, which was 3.45 U/mg higher than that of TZCR. The SOD activity of TZCR was the highest at 5 DPA, which were increased continuously after the anthesis, reached a maximum, and declined thereafter. The SOD activity of TZCR was 0.73-1.52 U/mg higher than that of TZWR at 10-20 DPA (Fig. 6A). The SOD activity of weedy rice and cultivated rice in Yangzhou showed a downward trend, but the SOD activity of YZWR was 0.99-1.96 U/mg signi cantly lower than that of YZCR at 3, 25 and 30 DPA (Fig. 6C). SOD activity of weedy rice and cultivated rice was higher from Dandong at 3 to 10 DPA, and there was no signi cant difference between them. The SOD activity decreased at 15 to 30 DPA, but the decline rate of weedy rice was faster, which was signi cantly lower than that of cultivated rice by 1.25-2.50 U/mg (Fig. 6D).
The POD activity of weedy rice in Taizhou showed a downward trend, the highest at 3 DPA, and was 184.53 U/g higher than that of TZCR, the POD activity of TZCR reached the maximum at 15 DPA, and declined thereafter. The POD activity of TZCR was 357.52-559.19 U/g higher than that of TZWR at 15-30 DPA (Fig. 7A). The POD activity of YZWR and YZCR showed a downward trend, but the decline rate of YZWR was slower. The POD activity of YZWR was 103.25 U/g lower than that of YZCR at 5 DPA, and was signi cantly higher than that of cultivated rice by 218.56 U/g at 20 DPA (Fig. 7B). The POD activity of MMWR reached the highest at 5 DPA, which was 219.77 U/g higher than that of MMCR, while the POD activity of MMCR reached the highest at 3 DPA, the POD activity of MMCR was 154.35 U/g higher than that of MMWR at 20 DPA (Fig. 7C). There was no signi cant difference in POD activity between weedy rice and cultivated rice in Dandong (Fig. 7D).  2018) found that cell viability of endosperm directly related to PCD, endosperm cells exhibited deformed nuclei and a loss of membrane integrity during early wheat grain lling. In current research, it was found that PCD occurred in endosperm cells of weedy rice and cultivated rice at the early stage of grain lling, and the process of PCD was basically completed in endosperm tissues at the late lling stage (Figs. 1-4). The endosperm of weedy rice and cultivated rice still maintained dehydrogenase activity and cell activity after nuclear disintegration (Figs. 1-4), which was consistent with previous studies. However, compared with cultivated rice, the nucleus of endosperm cells in weedy rice was deformed and disintegrated earlier than that in cultivated rice, and endosperm cells of weedy rice lost activity earlier than cultivated rice. This implied that the process of PCD in endosperm cells of weedy rice was faster than that of cultivated rice, and this may be one of the important physiological mechanisms of early maturity in weedy rice. Plant endogenous hormones are closely related to the process of PCD, altering endogenous hormone concentrations during grain lling could delay endosperm PCD, increasing grain lling time, such as abscisic acid (ABA), ethylene, and gibberellic acid (GA) can regulate PCD in the developing endosperm Gallie 1999, 2000;Li et al. 2018). However, the difference of hormone content between weedy rice and cultivated rice and its relationship with PCD during grain lling need to be further studied.

Discussion
The occurrence of PCD in plant cells is mainly caused by the accumulation of reactive oxygen species (Breusegem and Dat 2006), anti-oxidative enzymes system such as SOD, POD and CAT can protect cells by scavenging reactive oxygen species, and their activities are closely related to plant anti-aging (Corpas et al. 2006). SOD as the rst enzyme involved in the scavenging reaction of reactive oxygen species, can catalyze the disproportionation of superoxide to produce H 2 O 2 , while CAT and POD can transform H 2 O 2 into water and oxygen (Corpas et al. 2006). The activities of SOD and CAT were higher in the grain during rice endosperm development (Lan et al. 2004). We found that at least one antioxidant enzyme activity of weedy rice was lower than that of associated cultivated rice. Anti-oxidative enzymes activity is closely related to rice nature senescence and maturity. It has been reported that compared with rice varieties with a longer growth period, the activities of CAT and POD in leaves of rice varieties with a shorter growth period were lower, and senescence earlier (Wang et al. 2010). Therefore, we speculate that the rapid PCD process in the endosperm of weedy rice may be closely related to the activity of antioxidant enzymes. Under low antioxidant enzyme activity, cell can't effectively scavenge oxygen free radicals, cell macromolecules were poisoned, which accelerated the process of PCD in the endosperm of weedy rice. Compared with SOD and POD, CAT may play a more important role in scavenging reactive oxygen species . However, as the contents of reactive oxygen species and malondialdehyde (MDA) in endosperm of weedy rice and cultivated rice are not determined, the relationship between PCD process and ROS scavenging capacity of weedy rice and cultivated rice needs to be further veri ed. It has been reported that ascorbate peroxidase (APX), dehydroascorbic reductase (DHAR), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione (GSH), ascorbic acid (ASA) and other nonenzymatic substances can remove reactive oxygen species, which may play an important role in regulating grain lling and PCD process of endosperm cells (Yamauchi et al. 2001). The difference of these enzymes activity during endosperm development between weedy rice and cultivated rice will be the focus of the next study.
The PCD process is closely related to grain lling, and the factors affecting the grain lling can also affect the process of PCD.
To date, research on PCD process and grain lling differences in rice has mostly been based on enzyme activity, hormone balance, and PCD-related genes expression (Yang et al. 2003;Yin et al. 2012). Many studies have shown that starch synthase activity and hormone level are the main causes of differences in grain lling . Thirty-three major enzymes are reported to be involved in sucrose-to-starch conversion (SSC) during endosperm development in rice (Nakamura et al. 1989 play key roles in this process (Nakamura et al. 1989;Wang et al. 2015). Reports have shown that many genes are involved in controlling the process of SSC, including SuS2, SuS4, OsCIN2, OsINV2, AGPS1, AGPS2b, AGPL2, SSSIIa, SSSIIc, GBSSI, and SBEI . The relationship between activities of key enzymes and expressions of genes involved in sucrose-to-starch conversion and PCD need to be further studied. Plant hormones, such as ABA and ethylene, play vital roles in regulating grain lling. An appropriate level of ABA can enhance the key enzyme activity and gene expression related to starch metabolism and improve grain-lling rate . Ethylene can enhance the active oxygen system and stimulate free radical production in grains, and ethylene-induced H 2 O 2 can reduce grain weight and grain-lling rate Chen et al. 2013). Combined with this study, we can prolong the duration of PCD process and enhance the enzyme activities of antioxidant system by spraying some kind of chemical regulators, so that weedy rice has a longer lling period, which is later than cultivated rice. When the cultivated rice is mature and harvested, weedy rice is not mature, which eventually makes weedy rice di cult to disperse and spread, and nally control weedy rice. PCD is a physiological process determined by PCD related gene and plays an indispensable role in plant development (Schmid et al. 1999). In addition, the differences of PCD related genes (Os02g48450, Os04g02120, Os04g08390, Os05g31570, Os06g17970, Os08g30634, Os09g14410, Os09g30220, Os11g13940, Os11g38440, Os11g38580, and Os12g14330) (Yin et al. 2012), degradation of nuclear DNA and other indicators related to PCD process in endosperm cells between weedy rice and cultivated rice need to be further studied.

Conclusion
The endosperm cells of weedy rice degraded and lost viability earlier and more rapidly than those of ACR. The ability of scavenging reactive oxygen species by endosperm cells of weedy rice was weaker than that of ACR. The PCD process of endosperm cells in weedy rice was faster than that in cultivated rice. The rapid PCD process shortened the grain lling period of weedy rice, and eventually led to the early maturity of weedy rice. A better understanding of the mechanisms involved in PCD process of endosperm cells will improve the design of management strategies for weedy rice.     Changes in activities of POD in weedy rice and cultivated rice. A, weedy and cultivated rice from Taizhou; B, weedy and cultivated rice from Yangzhou; C, weedy and cultivated rice from Maoming; D, weedy and cultivated rice from Dandong. TZWR: weedy rice from Taizhou; TZCR: cultivated rice from Taizhou; YZWR: weedy rice from Yangzhou; YZCR: cultivated rice from Yangzhou; MMWR: weedy rice from Maoming; MMCR: cultivated rice from Maoming; DDWR: weedy rice from Dandong; DDCR: cultivated rice from Dandong.