A High Temperature-Dependent Mitochondrial Lipase EXTRA GLUME1 Promotes Floral Phenotypic Robustness against Temperature Fluctuation in Rice (Oryza sativa L.)

The sessile plants have evolved diverse intrinsic mechanisms to control their proper development under variable environments. In contrast to plastic vegetative development, reproductive traits like floral identity often show phenotypic robustness against environmental variations. However, it remains obscure about the molecular basis of this phenotypic robustness. In this study, we found that eg1 (extra glume1) mutants of rice (Oryza savita L.) showed floral phenotypic variations in different growth locations resulting in a breakdown of floral identity robustness. Physiological and biochemical analyses showed that EG1 encodes a predominantly mitochondria-localized functional lipase and functions in a high temperature-dependent manner. Furthermore, we found that numerous environmentally responsive genes including many floral identity genes are transcriptionally repressed in eg1 mutants and OsMADS1, OsMADS6 and OsG1 genetically act downstream of EG1 to maintain floral robustness. Collectively, our results demonstrate that EG1 promotes floral robustness against temperature fluctuation by safeguarding the expression of floral identify genes through a high temperature-dependent mitochondrial lipid pathway and uncovers a novel mechanistic insight into floral developmental control.


Introduction
The sessile plants have evolved various exquisite adaptive strategies to cope with environmental changes [1,2]. Among them, phenotypic plasticity is the ability of a single genotype capable of producing different phenotypes in response to varying environments [3][4][5][6]. For an integral high fitness, morphologies of vegetative organs of a single plant, such as roots, leaves and stems, require a high phenotypic plasticity [7][8][9][10], whereas that of reproductive organs, such as flowers, fruits and seeds, are always associated with low plasticity also known as phenotypic robustness/stability [11][12][13][14][15]. Thus, plants must coordinate the developments of these organs.
Compared with progresses in understanding the molecular mechanisms of high phenotypic plasticity [10,[16][17][18][19], very little is known about the molecular basis of phenotypic robustness [20,21]. Recent studies have shown that there are a group of specific genes regulating the degree of phenotypic plasticity and determining the reaction norm of a trait among various environments, which are termed plasticity genes [22][23][24]. However, most of the identified plasticity genes are high plasticity-associated [16,[25][26][27], only few promote phenotypic robustness [28][29][30]. Members of HSP90 (HEAT SHOCK PROTEIN 90) family, as central hubs of numerous biological pathways, are required for maintenance of phenotypic robustness in both animals and plants [28,[31][32][33][34]. MSH1 (MutS HOMOLOG1), a homolog of bacterial mismatch repair protein MutS, has been reported to repress the developmental plasticity of plant architecture, leaf morphology and flowering time in several dicot and monocot plants [29,35]. A nuclear protein RPL1 (RICE PLASTICITY1) in rice also appears to promote the relatively stable plant architecture and panicle structure between different environments [30]. Despite these discoveries, we still know very little about the molecular mechanisms of phenotypic robustness, especially that of plant reproductive traits. In addition to the known epigenetics-dependent transcriptional regulation and hormone signaling [20,[29][30][31]35], lipid homeostasis is also known to influence phenotypic robustness [36,37]. Coordinated regulations of cellular lipid homeostasis are crucial to organisms' adaptive robustness under severe temperatures [37][38][39][40]. Furthermore, lipid-related synthetases and lipases can also be regulated at transcriptional and posttranslational levels to influence lipid homeostasis [41,42]. Among them, mitochondriaassociated lipid metabolism is key to the lipid homeostasis [43]. For instance, Arabidopsis seedlings with decreased cardiolipin in mitochondrial membrane are easier to turn yellow and necrotic under extended darkness or heat due to a failure of mitochondrial morphogenesis, showing a lowered stability [41,44,45]. However, it remains unclear whether mitochondria also mediate the phenotypic robustness in plant reproductive organs.
Flower morphology, as a gold standard in plant taxonomy, has the most remarkable robustness within and between individuals of the same population [11,46], making it an ideal trait for studies on the molecular basis of phenotypic robustness against environmental fluctuation. Nevertheless, so far no gene has been identified to regulate the phenotypic robustness of floral identity, although several environment-dependent floral mutants have been reported [47][48][49][50][51][52]. We previously found that a rice floral mutant eg1 (extra glume1) exhibited a floral variation in different growth conditions [53], implying that EG1 is likely involved in floral robustness. Recent studies have shown that EG1 encodes a putative lipase regulating rice floral identity and meristem determinacy [53]. It also functions in JA (jasmonic acid) biosynthesis to promote the expression of floral identity gene OsMADS1 through an EG2/OsJAZ1, OsCOIb and OsMYC2mediated JA signaling pathway [54], similar to its homologous genes AtDAD1 (DEFECTIVE IN ANTHER DEHISCENCE1) and AtDGL (DONGLE) in Arabidopsis [55,56]. In this study, we find that EG1 is a predominantly mitochondria-localized functional lipase and promotes floral robustness against temperature fluctuation in a high temperature-dependent manner. Collectively, our results reveal a novel molecular mechanism underlying floral phenotypic robustness.
Results eg1 shows high plasticity in floral identity through an interaction of genotype and environment Previously, we found that eg1 displayed a floral identity variation possibly influenced by growth conditions [53]. To examine if this variability was mainly due to the environmental alterations, we analyzed the spikelet phenotypes of eg1-1 (in indica ZF802 background) and eg1-2 (in japonica ZH11 background) in two groups of separate environments (Fig 1) and found that the floral phenotypic variability of eg1 is likely caused by both genotype and environment. To define the phenotypic variability, we divided the spikelet phenotypes of eg1 into six groups, which were called variable phenotypes, including Wl (WT-like), eg (extra glume), pl (palea to lemma), sp (smaller pa), le (long empty glumes) and rs (reiterated spikelets) (Fig 1A, S1 Fig and S1 Table). The results showed that sp and rs of eg1-1 as well as Wl, le, eg and sp of eg1-2 exhibited significant plasticity between two environments, especially le of eg1-2, which displayed the highest plasticity ( Fig 1B and 1C), suggesting that environment also contributes to the variations of floral phenotypes of eg1. To further examine the relationships of genotype (G), environment (E), genotype-environment interaction (GxE) and the variable phenotypes, we calculated their effects on phenotypes by two-factor ANOVA and found that Wl and pl were affected mainly by G, eg by both E and G but rarely by GxE, sp, le and rs by all three factors, and among them, le could serve as a marker for the phenotypic plasticity of eg1-2 due to its large proportion in a panicle and opposite phenotypes between two environments ( Fig 1A). Taken together, these results showed that eg1 shows higher floral plasticity, suggesting that EG1 promotes the floral robustness in rice.
Allelic eg1 and their genetic backgrounds together regulate the eg1 floral plasticity To further examine the influence of genotype on the floral plasticity of eg1, we swapped the genetic backgrounds of two eg1 alleles. eg1-1 in a largely japonica background showed high phenotypic plasticity especially for le and rs phenotypes, similar to eg1-2 (ZH11), whereas eg1-2 in an indica-dominant background showed low floral plasticity similar to eg1-1 (ZF802) (Fig  2A), indicating that genetic backgrounds also influence the phenotypic plasticity of eg1 spikelets.
To verify this finding, we further used CRISPR/Cas9 technology to construct eg1 alleles in Nipponbare (japonica) and Dular (indica) backgrounds. Two types of spikelet phenotypes were found in eg1-4 allele with Dular background and both showed low plasticity (Fig 2B), while eg1-5 and -6 alleles in Nipponbare background showed relatively higher plasticity than Floral structures and two-factor plots with percentages per panicle in the y axis of all variable phenotypes are shown in the left and middle columns, respectively. Effects of G, E and GxE on each variable phenotype shown in the pie charts (right column) were analyzed by twoway ANOVA. Bar = 2 mm in floral structures. (b) Statistical analysis of the phenotypic plasticity of eg1 and wild-type spikelets in two different planting locations. Beijing, Beijing summer. Lingshui, Lingshui winter. (c) Statistical analysis of the phenotypic plasticity of eg1 and wild-type spikelet in two different planting seasons. LS Feb, Lingshui Feb. LS Apr, Lingshui Apr. Wl, WT-like; eg, extra glume; pl, palea to lemma; sp, smaller pa; le, long empty glumes; rs, reiterated spikelets. Values are means ± SE, number of analyzed panicles ! 20 in (b), ! 10 in (c). Significant difference was determined by ANOVA, *P < 0.05, **P < 0.01. doi:10.1371/journal.pgen.1006152.g001 alleles in two indica backgrounds ZF802 and Dular, similar to that in japonica ZH11 (Fig 2C  and S2 Fig), suggesting that the floral plasticity of eg1 alleles in japonica backgrounds tend to be higher than that in indica backgrounds. In another aspect, eg1 alleles in indica backgrounds had severer floral disturbance than that in japonica concerning Wl and rs phenotypes (Fig 2  and S1 Fig), suggesting that EG1 has functions in both floral robustness and identity, which are  Table, and percentages of them in a panicle are shown in the y axis. le, lemma; pa, palea; st, stamen; eg, empty glume; if, inflorescence primordia; sp, smaller pa; lel, lemma-like organ; pl, palea-lemma mosaic organ. Bars = 2 mm. Values are means ± SE, number of analyzed panicles ! 5, and significant difference was determined by ANOVA, *P < 0.05, **P < 0.01. differentiated in two subspecies. To explore the possible causes of these differentiation, we compared the cis-elements and expressional level of EG1 in several japonica and indica varieties and discovered the correlative differences in both cis-elements and expressional levels between japonica and indica (two types) varieties (S3 Fig and S2 Table), which implied that transcriptional differences may be a crucial cause of functional differentiation of EG1 in subspecies. All these results indicated that both eg1 allelic variations and their genetic backgrounds regulate the floral plasticity of eg1.
Temperature is a major environmental factor mediating the plastic development of eg1 spikelets In order to find out the environmental factors mediating the plastic development of eg1 spikelets, we first compared the growing conditions for phenotypic analysis and found a marked difference in daily high temperatures of two environments (S4 Fig), suggesting that the temperature variation between two environments could be a major environmental factor influencing the eg1 plasticity. To verify this prediction, floral plasticity of wild-types and eg1 alleles were examined in two artificial growth chambers with 35°C light 12 hr / 20°C dark 12 hr and 25°C light 12 hr / 20°C dark 12 hr respectively, while other growth conditions were kept identical. The low plasticity of eg1-1 and nearly 70% le phenotypes of eg1-2 showed that the floral plasticity of eg1 in the chambers was similar to and even higher than that under natural growth conditions (Fig 3). These results showed that temperature is a major environmental factor mediating the floral plasticity of eg1.

EG1 encodes a predominately mitochondria-localized functional lipase
Previously, EG1 was shown to be localized in chloroplasts in transient expression assays [54]. However, EG1-like lipases appear to have variable subcellular locations [56][57][58][59]. To examine the subcellular localization of EG1 in vivo, two different EG1 and GFP fusion proteins driven by 35S promoter were first expressed in rice protoplasts and were found to be co-localized with both mitochondrial specific dye Mito Tracker Red and mitochondrial maker protein MTS-mOrange [60] but hardly with chloroplast auto-fluorescence, and an EG1-GFP fusion protein driven by native promoter was also detected in mitochondria (Fig 4A and 4B), suggesting that EG1 protein is mainly, if not all, localized in mitochondria. To compare this finding with the previous one, the reported vector pCAMBIA1301-Pro35S:EG1-GFP [54] was also examined in our transient system and a similar localization was detected (S5 Fig). To further verify the EG1 localization, subcellular fractionations of one-week seedlings of eg1-2 complementation lines with FLAG-EG1 (S6 Fig) were successively carried out and the EG1 fusion protein was predominately co-fractionated with mitochondria ( Fig 4D), confirming the mitochondrial localization of EG1 in vivo. Taken together, these results showed that EG1 encodes a protein predominately localized in mitochondria. To explore the biochemical function of EG1, we tested its lipase activity [53] in vitro and found that both the full-length EG1 and truncated EG1 without predicted targeting peptides showed significant lipase activity (Fig 4E), indicating that EG1 encodes a functional lipase. Taken together, our results showed that EG1 functions as a predominately mitochondria-localized lipase.
EG1 functions in a high temperature-dependent manner in the floral robustness regulation The dependence of the eg1 floral plasticity on environmental temperature raised a possibility that either EG1 or its product or both are likewise regulated by temperature. To examine these possibilities, some heat/cold responsive cis-elements were discovered in the 2 kb genomic sequence upstream of the start codon of EG1 (S3 Table), implying that its expression could be induced by extreme temperatures. To examine this possibility, one-week wild-type seedlings were treated under different temperatures and the EG1 transcript was found to accumulate gradually, to an extremely high extent under heat shock (42°C) as well as usual high temperature 35°C for rice ( Fig 5A) Table. le, lemma; pa, palea; eg, empty glume; if, inflorescence primordia; lel, lemma-like organ; pl, palea-lemma mosaic organ; leg, long empty glume in spikelet structures. Bars = 2 mm. Values are means ± SE, number of analyzed panicles >10, and significant difference was determined by ANOVA, *P < 0.05, **P < 0.01.   Values are means ± SE (n = 3), and significant difference was determined by ANOVA, *P < 0.05, **P < 0.01, and rice α-TUBULIN as the reference. (b) Western blot analysis of FLAG-EG1 protein accumulation under different temperatures and different tissues in the EG1 complementation lines for 24 hr. Cp, Complementation lines; Ct, non-transgenic wild-type control. HC, Heavy chain of IgG; NS, Nonspecific band (as a loading control). (c) Temperature-dependent lipase activity of EG1. EG1 (Full) and EG1 (Δ45) respectively refer to full-length and no N-terminal (45 AA) protein of EG1 fused to SUMO peptide. Values are means ± SE for three independent experiments. (d) Floral phenotypes of eg1 mutants in a condition of 40°C light 12 hr / 30°C dark 12 hr. Spikelets of eg1-1 with pl, eg and rs phenotypes are shown on i, ii and iii, respectively. Spikelets of eg1-2 with eg and pl phenotypes are shown on iv and v, and with multilayer lemma-like glumes (lel) and/or undetermined inflorescences primordia are on vi to x. x is the inside of ix. le, lemma; pl, palea-lemma mosaic organ; eg, empty glume; lel, lemma-like organ; pa, palea; st, stamen; if, inflorescence primordia. Bars = 2 mm. doi:10.1371/journal.pgen.1006152.g005 extreme high temperature 42°C than 25°C and 35°C (Fig 5B and S7D Fig), indicating a stabilization of EG1 protein under heat stress. Furthermore, we detected that lipase activity of EG1 fusion proteins increase as temperature rising (Fig 5C), consistent with the assumption of EG1's function required under high temperatures. Additionally, we also examined the effect of high temperatures on EG1 subcellular localization, and found no obvious translocation in protoplast system (S8A Fig), while failed to detect EG1 protein in the subcellular fractions of complementation lines except under heat stress for its minute amount (S8B Fig), suggesting that temperature does not significantly influence the subcellular localization of EG1. The increased the transcriptional level, protein stability and lipase activity of EG1 under high temperatures, implying its more significant role under high temperatures. To verify this hypothesis, we observed the floral phenotypes of eg1 mutants under extremely high temperatures and found much severer spikelets in eg1 mutants especially in eg1-2, with multilayer lemma-like organs and undetermined inflorescence meristem, which have never been found in other temperature conditions (Fig 5D), showing a more significant function of EG1 at higher temperatures in floral robustness. eg1 was also found to grow faster than wild-type during primary growing days [54], and we detected this phenotype was much severer under extremely high temperature than others compared with wild-type, which was consistent with the floral phenotype (S9 Fig). Therefore, we concluded that EG1 functions in a high temperature-dependent manner to regulate the floral robustness.

EG1 mediates the transcriptional responses of downstream genes and pathways to environmental fluctuation
Since the floral plasticity of eg1 was influenced by both genotype and environment, to examine the molecular mechanism of genotype-environment interaction in floral plasticity, transcriptomes of inflorescences of two eg1 alleles (eg1-1 and eg1-2) and their wild-types (ZF802 and ZH11) in Beijing and Lingshui were analyzed. First, to evaluate the reliability of the transcriptomic data, we divided all transcripts into 33 modules by co-expression network analysis and analyzed their correlations with six variable phenotypes, and it turned out that the relationships among the variable phenotypes derived from these correlations were quite similar to their morphological correlations (S10 Fig), showing a good reliability of the transcriptomic data. Second, through overall comparisons of all transcriptomes, we discovered that the expression patterns of floral transcriptomes of eg1 alleles between two environments were significantly different from their wild-types (S11A Fig), indicating a role of EG1 in regulating expressions of environmentally responsive genes. The numbers of environmentally responsive genes in eg1 mutants were much larger than that of wild-types, in contrast to the similar numbers between two wild-types or two eg1 alleles (Fig 6A and S11B Fig), implying that EG1 negatively regulates the responses of its downstream genes to environment. To verify this finding, we analyzed the effects of G, E, and GxE on transcriptomes of eg1 and wild-type by two-way ANOVA and found that the number of genes significantly affected by E and GxE in eg1 were significantly larger than that in wild-types (Fig 6B, S11C Fig and S4 Table), displaying a switch of many genes from G-affected to E/GxE-affected ones (Fig 6C), indicating that EG1 represses its downstream genes not only to respond to, but also to interact with environment. Furthermore, the effects of the three factors on several important pathways varied significantly between eg1 and wild-type, including the pathways related to temperature response, lipid metabolism and floral development (Fig 6D and S5 Table), indicating that EG1 mediates a crosstalk of these pathways with environment. Since EG1 was reported to regulate JA biosynthesis [54], in order to analyze its effect on the floral robustness control, we examined the expressional patterns of JA biosynthesis and signaling associated genes in our transcriptome  , and found that the transcriptional responsive patterns to environment or transcriptional level of several JA signaling genes (JAZ7 and JAZ8) and JA biosynthesis genes (four methyltransferase genes) are varied in eg1 mutants (S12 Fig), implying a possible role of JA in the EG1-associated floral robustness regulation but different from previously reported [54]. Taken together, these results revealed that EG1 mediates the transcriptional responses of downstream genes and pathways to environmental fluctuation.
OsMADS1, OsMADS6 and OsG1 act downstream of EG1 to mediate floral robustness regulation The significant transcriptional effects on the floral development pathways based on the G, E and GxE analysis in eg1 mutant suggested that floral identity genes are likely involved in EG1dependent floral robustness regulation. To examine this, expressional variation of thirteen floral identity genes between two environments were further analyzed, and seven (OsMADS1, OsMADS6, OsG1, OsMADS4, OsMADS7, OsMADS8, OsMADS58) of them showed both varied environment-dependent expression patterns and repressed expression levels in eg1 (S13 Fig), indicating their positive regulatory roles in the floral robustness. To further examine this possibility, genetic analysis between EG1 and three of them (OsMADS1, OsMADS6 and OsG1) were performed. OsMADS1 and OsMADS6 are two major genes regulating glume identity and floral determinacy in rice [61][62][63][64][65][66][67][68][69], and their expressions were significantly varied in eg1 (Fig 7E and  S13 Fig), indicating that EG1 is required for their expressions. To examine their genetic relationships with EG1, a double mutant of OsMADS1 mutant allele nsr [61] and eg1-1 was generated and it exhibited longer and leafy lemmas/paleas similar to nsr, with all inner three whorls replaced by half-developed inflorescences or inflorescence primordia, which is severer than both single mutants (Fig 7B), indicating that OsMADS1 functions downstream of EG1 in lemma/palea identity and they may together regulate the determinacy of inner three whorls. In addition, the double mutant of eg1-1 and OsMADS6 mutant allele osmads6-5 [66] showed abnormal paleas, with all transformed into one or two small lemma-like glumes and mostly with inflorescence primordia inside the spikelets (Fig 7C), indicating that OsMADS6 functions downstream of EG1 in specifying palea but may also regulate floral determinacy together with EG1. osmads6-5 exhibited weaker floral disturbance in the F2 population when crossed with eg1-1 (ZF802), with lemma-palea mosaic paleas and usually normal inner whorls (Fig 7C), showing its floral phenotype is also greatly influenced by genetic backgrounds. To further examine the relationships among EG1, OsMADS1 and OsMADS6 especially in floral determinacy, nsr osmads6-5 and eg1-1 nsr osmads6-5 were successively generated. The floral meristems of these two mutants similarly generated continuous glume primordia or became inactive before inner three whorls developed (Fig 7D), which were more dedifferentiated than the inflorescence primordia of eg1-1 nsr and eg1-1 osmads6-5, supporting the findings that both OsMADS1 and OsMADS6 act downstream of EG1 and they together control the floral differentiation of inner three whorls. Compared with eg1-1, the rs plasticity of eg1-1 nsr osmads6-5 totally disappeared when these two MADS genes were both deficient (Fig 7B and 7D), supporting the important roles of OsMADS1 and OsMADS6 in the rs plasticity regulation. Additionally, the spikelet phenotype of eg1-1 nsr [54] and nsr osmads6-5 [68,69] were consistent to the previously reported, and eg1 was linked with the lemma-like (lel) structure, and not affected by the deficiency of OsMADS1 and OsMADS6 (Fig 7b-7d), implying that it is probably a special in boxes indicate -log10 (P-value) of the pathway enrichment tested by Fisher's exact test with Bonferroni correction and blank boxes no statistical significance. Furthermore, the le phenotype (long empty glume) of eg1-2 has the highest plasticity among all variable phenotypes (see , and OsG1 (Long Sterile Lemma) and OsMADS19/34 are two crucial genes suppressing the elongation of empty glumes in rice [70][71][72]. The expression levels and patterns of OsG1 but not OsMADS19/34 appeared to be aberrant in eg1-2 ( Fig 7E and S13 Fig), implying that OsG1 may contribute to the le phenotype of eg1-2.
To confirm this, g1-ele allele of OsG1 [72] was used to obtain a double mutant by crossing with eg1-2, and it turned out that almost all empty glumes of eg1-2 g1-ele were elongated to lemmalike organs similar to g1-ele, exhibiting much lower plasticity compared with eg1-2 ( Fig 7F and  7G), indicating that OsG1 functions downstream of EG1 in regulating empty glume development and contributes to the plastic le phenotype of eg1-2. Taken together, these results show that OsMADS1, OsMADS6 and OsG1 all act downstream of EG1 to mediate the floral robustness regulation.

EG1 is a plasticity gene regulating the floral robustness against environmental fluctuation in rice
To our knowledge, no plasticity genes have been confirmed to function in floral robustness in flowering plants [47][48][49][50][51][52]. Given the sessile nature of plant species, uncovering this class of genes and dissecting their molecular mechanisms are crucial for understanding the biology of flower development and evolution. In this study, we have shown that EG1 encodes a mitochondria-localized lipase functioning as a plasticity gene to regulate the rice floral robustness by a coordinated transcriptional regulation of temperature, lipid metabolism and flower development pathways. First, eg1 alleles showed floral variations under both natural and artificial conditions and five eg1 alleles produced increased floral plasticity. Second, RNA expression, protein stability and lipase activity of EG1 can respond to environment enhancing its function significantly in severe conditions such as heat stress. Third, EG1 appears to possess a "buffering" function of repressing environmental stimuli to interfere the target genes, and when environmental pressure becomes severer such as heat stress, the strengthened EG1 function induced by heat is enough to buffer the stronger and more deleterious effect of heat on the responsive transcriptional pathways. Last, EG1 influences the expression of numerous floral identity genes, which are the direct contributors of plastic development of eg1 spikelets. Thus, EG1 is the first plasticity gene regulating plant floral robustness against environmental fluctuation. Our finding indicates that flowers retain a system containing EG1 to sense and respond to environmental stimuli to maintain its stable development, and suggests a mechanism of transition from high plasticity to robustness in flower by recruiting plasticity-repressing genes, which ensures the coordinated development of organs with different plasticity in one organism. eg1-1 and nsr mads6-5. Photos of nsr mads6-5 and eg1-1 nsr mads6-5 are shown as (b). Arrows on iii of nsr mads6-5 and eg1-1 nsr mads6-5 indicate the inactive growing points. (e) Relative expression levels of OsMADS1, OsMADS6 and OsG1 in wild-type and eg1 inflorescences of different growth stages by RT-qPCR. Results are means ± SE. Rice α-TUBULIN was used as the reference. (f) Genetic analysis of eg1 and g1-ele. Empty glume phenotypes of eg1-2 in Lingshui, g1-ele and eg1-2 g1-ele in Beijing/Lingshui are shown respectively. (g) The phenotypic plasticity of le in eg1-2, g1-ele and eg1-2 g1-ele. Values are means ± SE, number of analyzed panicles ! 5. le, lemma; pa, palea; st, stamen; pi, pistil; eg, empty glume; ca, carpel; fm, floral meristem; im, inflorescence meristem; if, inflorescence primordia; lel, lemma-like organ; gl, glume-like organ; lp, lemma-palea mosaic organ; log, lodicule-glume mosaic organ. leg, long empty glume. Bars = 2 mm, 200 μm and 200 μm in spikelet structures, SEMs and paraffin transverse sections respectively, except for 0.5 mm in ii of (d). doi:10.1371/journal.pgen.1006152.g007 Mitochondria could serve as a hub for the lipid metabolism mediatedfloral developmental robustness Lipid metabolisms are known to be involved in adaptive plasticity of organisms [36,37,41,42], and mitochondria, as the energy factory of eukaryotes and one of subcellular compartmentations of lipid metabolism, have been reported to be crucial to the adaptive stability of plant vegetative traits [41,43,44]. As EG1 is a predominately mitochondria-localized functional lipase, our results suggest that the mitochondria-mediated lipid metabolism plays an important role in the regulation of floral robustness against temperature fluctuation. However, it remains unclear how this could be carried out. It is likely that mitochondria-related lipid homeostasis could serve as a "buffer" to relieve the effect of environmental stimuli and mediate the temperature-dependent floral plasticity regulation (Fig 8). Recently, EG1 has been shown to influence JA synthesis, and JA signaling pathway regulates the transcription of floral identity gene OsMADS1 [54], showing that JA potentially mediates the crosstalk of EG1 and flower development related transcriptional factors. In fact, the most homologous gene of EG1 in Arabidopsis DGL has been verified to function in JA biosynthesis [55,56,73], though its chloroplast location was questioned [55], suggesting that a non-chloroplast localized lipase is likely to function in JA production (Fig 8). On the other side, we may fail to detect EG1 protein due to its minute amounts in chloroplasts. Furthermore, according to the much severer phenotype of eg1 and eg2-1D (a mutant allele of EG2/OsJAZ1) double mutant compared with two single [54], we noticed that JA signaling may be not the only pathway activated by EG1 to mediate the signal transduction from outside to inside of nucleus in the EG1-associated floral regulation, other lipid-related pathways and regulatory mechanisms are also possible (Fig 8).

Functional divergence of EG1 and its homologs in angiosperms
In our study, eg1 alleles showed a higher floral plasticity in japonica than that in indica varieties, while a severer floral disturbance in indica, revealing a functional divergence of EG1 in rice subspecies. No differences in coding sequences but cis-elements of EG1 in two subspecies were detected (S14 Fig), implying that this subspecific variation might be caused by an unknown differential responsive capability of promoters to environmental or endogenous stimuli. Besides, since genetic backgrounds are known to influence developmental outcomes via phenotypic modifiers [74,75], there also may be some indica-/japonica-specific modifiers to modulate EG1 function due to their different genetic backgrounds, which can be either epistatic genes or specific lipid substrates of EG1 required for floral developmental robustness. Genetic dissection of these subspecific modifiers of plasticity will provide further insights into the molecular mechanism of floral development. So far, all reported EG1 homologs in dicots have no apparent plasticity function in flowers [56][57][58][59], and EG1 homologs in monocots can be mainly divided into two clades, one similar to that in dicots and the other unique to monocot species based on the phylogenetic tree and predicted subcellular localizations (S13 Fig), we thus speculate that EG1 may have acquired a monocot-specific neofunctionalization in promoting floral robustness. Detailed genetic and biochemical analyses of these genes would provide additional clues to when and how the plasticity function and the subspecific divergence of EG1 arose in monocot species.
In conclusion, our results revealed a novel function of EG1 in floral developmental robustness against environmental fluctuation by mediating the mitochondrial lipid metabolism. Our finding also provide a genetic means to maintain the stable flower development under environmental stress ensuring grain yield stability in rice and potentially in other monocot species. Further studies should unlock the molecular crosstalk between mitochondria and nucleus in regulating floral developmental robustness.
Spikelets of eg1 were divided according to phenotypes of outer glumes (lemma, palea, empty glumes and extra glumes) following two rules: (1) the most significant mutant phenotype of eg1 is in the outer glumes; (2) outer glumes have linkages with inner organs: normal palea always linked with normal inner organs, lemma-like palea with increased stamens and pistils, smaller palea with decreased stamens, multilayer glumes linked with rs. Main panicles/inflorescences of plants at booting stage were used for phenotypic analysis. Percentages of all variable phenotypes in one panicle were calculated for comparisons, and in some cases more than one phenotype appeared in a single spikelet. Data were statistically analyzed by using one/two-way ANOVA (Excel). Effects of genotype, environment and genotype-environment interaction on all variable phenotypes were calculated using phenotypic statistic data of eg1 and wild-type grown in Beijing summer and Lingshui winter by two-way ANOVA. In CRISPR experiment, more than six independently homozygous lines were generated in each background, and their phenotypes were quite similar, thus only one/two lines were statistically analyzed and shown in our results.

Protein subcellular localization analyses
Full-length EG1 CDS was inserted into N-or C-terminal of GFP sequence of vector pBI221-35SPro:GFP, and full-length EG1 CDS with 1.5 kb native promoter into pCAMBIA1301-GFP. MTS-mOrange and Pro35S:COX11-GFP plasmids were kindly provided by Dr. Yaoguang Liu [60], and the reported vector pCAMBIA1301-35SPro:EG1-GFP was provided by Dr. Dabing Zhang [54]. All plasmids of high quality were prepared for protoplast transfection. Rice protoplast preparation from 2-week-seedlings grown in light and polyethylene glycol (PEG)-mediated transfections were performed as described by Bart et al. [78]. Images were captured by a confocal microscope (FluoView 1000, Olympus).

Lipase activity analysis in vitro
Full-length EG1 CDS and a truncated EG1 CDS without predicted targeting sequence (135 bp) with engineered N-terminal SUMO tag were separately cloned into pET-30a (Novagen) to generate His6-SUMO-tagged fusion proteins. Mitochondrial targeting sequence was predicted with MitoProt II online [80]. DGL CDS without targeting sequence [81] was introduced into vector PMAL-C2X (NEB). All the fusion proteins were expressed in E. coli BL21 (DE3). The His 6 -SUMO-tagged fusion proteins were purified using Ni-NTA (Novagen) and eluted with buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl and 250 mM imidazole. The MBP-tagged fusion proteins were purified using amylose resin (NEB) and eluted with buffer containing 50 mM Tris-HCl (pH 7.4) and 10 mM amylose. Imidazole in protein solutions was removed with desalting columns (Thermo Scientific) before lipase activity analysis.
p-nPB (p-nitrophenyl butyrate, Sigma) was used as the substrate of lipase analysis in vitro. Colorimetric assays for lipase activity of fusion proteins were performed as described by Seo et al. [82] with some modifications. A solution containing 1.11 mg/mL p-nPB (dissolved with isopropanol) and B solution containing 50 mM Tris-HCl (pH 7.4) and 0.1% Arabic gum were first prepared. Reactive solution was composed of 1 volume A solution and 9 volumes B solution with 2% Triton X-100. About 20 μl purified proteins (~5 μg) and 180 μl reactive solution were used for each reaction. After incubated under different temperatures for 30 min, p-nitrophenol formation from p-nitrophenyl butyrate was determined spectrophotometrically at 405 nm by an ELISA microplate reader. DGL and porcine pancreatic lipase (Sigma) were used for positive controls, and p-nitrophenol (Sigma) for the standard curve.

Physiological experiments
Cis-elements in the genomic sequence upstream of the start codon of EG1 was analyzed by PLACE online [83]. Wild-type ZF802, ZH11 and EG1 complementation lines were grown at 25°C for one week after germination, and wild-type ZH11 seedlings were grown in a consistent condition till 2 mm inflorescence meristem developed before being treated under different temperatures for different time. The transcripts of EG1 and FLAG-EG1 were analyzed by RT-QPCR or RT-PCR. The root and shoot phenotypes of seedlings were statistically analyzed after growing under consistent temperatures for six days after germination. Other condition parameters of physiological experiments were daylight 12 hr, light intensity 50 μmol m -2 s -1 and relative air humidity 60%. We use 42°C as an extremely high temperature for short treat but 40°C for long treat by considering the tolerance of plants.
FLAG-EG1 protein in EG1 complementation lines was enriched by immunoprecipitation for its small amount and detected by western blot with α-FLAG (Sigma).

Environmental transcriptome analysis
Two mm inflorescence meristems of eg1-1, eg1-2 and their wild-types ZF802 and ZH11 planted in Beijing summer and Lingshui winter were used for RNA sequencing, and two biological replicates were performed. Total RNAs were isolated with TRIzol kit (Invitrogen). Illumina sequencing libraries were prepared according to the manufacturer's instructions (Illumina Part # 15026495Rev. D), and sequenced with Illumina system Hiseq2500. Analysis of RNA-seq data was conducted following the standard protocol as described by Trapnell et al. [84]. The raw reads of RNA-seq were mapped to MSU_IRGP_V7 (japonica) and Oryza_indicaASM465 v1.23 (indica) by Tophat [85]. Cuffdiff [85,86] was used to identify the differentially expressed genes between different genotypes or different environments. Co-expression network analysis was performed using R packages WGCNA. Enrichment pathways of genes significantly and specifically affected by G, E or GxE was analyzed and the -log10 (P-values) was tested by Fisher's exact test with Bonferroni correction as described by Lu et al. [87].