Functional Genomics Laboratory, Institute of Molecular and Cell Biology, Proteos, Singapore 138673
Severe Arabidopsis (Arabidopsis thaliana) gibberellin (GA)-deficient mutant ga1-3 fails to germinate and is impaired in floralorgan development. In contrast, the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant confers GA-independent seed germination andfloral development. This fact suggests that GA-regulated transcriptomes for seed germination and floral development areDELLA dependent. However, it is currently not known if all GA-regulated genes are GA regulated in a DELLA-dependentfashion and if a similar set of DELLA-regulated genes is mobilized to repress both seed germination and floral development.Here, we compared the global gene expression patterns in the imbibed seeds and unopened flower buds of the ga1-3 mutantwith that of the wild type and of the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant. We found that about one-half of total GA-regulatedgenes are apparently regulated in a DELLA-dependent fashion, suggesting that there might be a DELLA-independent or-partially-dependent component of GA-dependent gene regulation. A cross-comparison based on gene identity revealed thatthe GA-regulated DELLA-dependent transcriptomes in the imbibed seeds and flower buds are distinct from each other.Detailed ontology analysis showed that, on one hand, DELLAs differentially regulate the expression of different individualmembers of a gene family to run similar biochemical pathways in seeds and flower. Meanwhile, DELLAs control manyfunctionally different genes to run specific pathways in seeds or flower buds to mark the two different developmentalprocesses. Our data shown here not only confirm many previous reports but also single out some novel aspects of DELLAfunctions that are instructive to our future research.
Plant development is an orderly process that startsfrom seed germination to juvenility, maturity, flower-ing, and fruiting. The whole process is modulated byphysical, chemical, and biological components in theenvironment as well as by several internal factors,including auxins, abscisic acid (ABA), cytokinins, eth-ylene, and GA. GA is essential for multiple processesof plant development, such as seed germination, stemelongation, and floral development (Richards et al.,2001; Olszewski et al., 2002; Peng and Harberd, 2002;Sun and Gubler, 2004). In Arabidopsis (Arabidopsisthaliana), the severe GA-deficient mutant ga1-3, whichcontains greatly reduced levels of bioactive GAs, isdefective in seed germination, retarded in vegetativegrowth, and impaired in the development of floralorgans (Koornneef and van der Veen, 1980; Wilsonet al., 1992; Sun and Kamiya, 1994).
In recent years, significant progress has been madeto understand the molecular mechanism of GA action.
In brief, the binding of GA to its soluble receptorGIBBERELLIN INSENSITIVE DWARF 1 (OsGID1) orOsGID1-like (Ueguchi-Tanaka et al., 2005; Hartweckand Olszewski, 2006) triggers the degradation of plantgrowth repressor DELLA proteins (DELLAs) via the26S proteasome pathway (Silverstone et al., 2001; Fuet al., 2002; Itoh et al., 2002; Hussain et al., 2005). Thedegradation process is mediated by the GA-specificF-box proteins OsGID2 (Sasaki et al., 2003) and AtSLY1(McGinnis et al., 2003; Dill et al., 2004; Fu et al., 2004).The degradation of DELLAs will release the plantsfrom the DELLA-mediated growth restraint (Harberd,2003). DELLAs are named after a highly conservedmotif at their N termini that is important for GAsensitivity (Peng et al., 1999; Boss and Thomas, 2002;Chandler et al., 2002), and they form a subfamily of theGRAS family of putative transcription regulators(Pysh et al., 1999; Richards et al., 2000). There arefive DELLAs in Arabidopsis: GAI, RGA, RGL1, RGL2,and RGL3 (Dill and Sun, 2001; Lee et al., 2002; Wen andChang, 2002; Hussain et al., 2005). Genetic studieshave revealed that GAI and RGA (Peng et al., 1997;Silverstone et al., 1998) are involved in repressing stemelongation since loss-of-function of both GAI and RGAcompletely suppressed the dwarf phenotype of ga1-3mutant (Dill and Sun, 2001; King et al., 2001). Duringfloral development, RGA, RGL2, and RGL1 jointlyrepress petal and stamen development. Combinationsof loss-of-function mutations of RGA, RGL1, and RGL2suppressed the male sterile phenotype of the ga1-3mutant (Cheng et al., 2004; Tyler et al., 2004; Yu et al.,
1 This work was supported by the Agency for Science, Technology,and Research in Singapore.
2 These authors contributed equally to the paper.* Corresponding author; e-mail [email protected]; fax
65–6779117.The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Jinrong Peng ([email protected]).
[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.106.082289
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2004). On the other hand, RGL2 is the key repressor ofseed germination and this function is enhanced byGAI and RGA (Lee et al., 2002; Tyler et al., 2004; Caoet al., 2005).
The fact that, in the absence of exogenous GA, ga1-3plants lacking the four DELLA proteins GAI, RGA,RGL1, and RGL2 (i.e. ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1mutant line) can germinate, bolt, and produce fullydeveloped fertile flowers as the wild-type controlsuggests that DELLAs are functional redundant fac-tors and they act as the central signaling molecules inGA-mediated seed germination, stem elongation, andfloral development pathways (Cheng et al., 2004; Tyleret al., 2004; Yu et al., 2004; Cao et al., 2005). However, asa group of putative transcription regulators (Pysh et al.,1999; Richards et al., 2000), the molecular mechanismof DELLAs repressing plant growth is largely un-known. For example, it is not known whether DELLAssimply control the expression of a similar set of genesto repress seed germination, stem elongation, andfloral development, or whether they mobilize differentsubsets of genes in the genome to modulate these dif-ferent processes. Meanwhile, it is of our great interestto know if all GA-regulated genes are GA regulated ina DELLA-dependent fashion.
One way to answer the above questions is to com-pare the gene expression patterns in the ga1-3 mutantto that in the plants of no DELLA activity in the ga1-3background. The ga1-3 mutant fails to germinate andis retarded in floral development, suggesting that thetranscriptome for germination and floral developmentin the ga1-3 mutant must be kept at a repressive state(Ogawa et al., 2003). On the other hand, the fact thatthe ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant confers GA-independent seed germination and flowering suggeststhat, in this mutant line, the transcriptomes responsi-ble for germination and floral development must havebeen constitutively activated. It is reasonable to spec-ulate that genes normally up-regulated by GA wouldexpress at lower levels in ga1-3, and the stabilized highlevels of DELLA repressors in ga1-3 would be respon-sible for a proportion of these lower expressed genes(Lee et al., 2002; Tyler et al., 2004). Therefore, the genesthat are genuinely repressed, directly or indirectly, byDELLAs will be restored to wild-type levels or evenhigher in the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutantbecause this mutant line is absent from four DELLAproteins. Vice versa, genes activated by DELLAs mightexpress at higher levels in ga1-3 and be brought back towild-type levels or even lower in the ga1-3 gai-t6 rga-t2rgl1-1 rgl2-1 mutant. Therefore, comparing the expres-sion profile in ga1-3 with that in the ga1-3 gai-t6 rga-t2rgl1-1 rgl2-1 mutant would help to identify the set ofDELLA-dependent transcriptomes essential for seedgermination and floral development. Because we alsowished to compare the gene expression in ga1-3 show-ing phenotypic suppression by the quadruple DELLAknockout to gene expression in the wild-type plants,we chose to compare wild type and ga1-3 instead of ga1-3treated with GA. We first identified GA-regulated
(both up- and down-regulated) transcriptomes in bothimbibed seeds and young flower buds by comparingthe expression patterns between the ga1-3 mutant andthe wild-type control. Then, we identified DELLA-dependent (both up- and down-regulated) transcrip-tomes by finding out the subgroup of GA-regulatedgenes with their expression restored to wild-type levelsin the ga1-3 rga-t2 gai-t6 rgl1-1 rgl2-1 mutant. Dataanalysis showed that, in both imbibed seeds and youngflower buds, approximately one-half of genes down- orup-regulated in ga1-3 were apparently restored to thewild-type level in the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1mutant, which suggests that (1) these GA-regulatedDELLA-dependent genes are likely responsible for medi-ating seed germination or floral development and (2)there might be a DELLA-independent or -partially-dependent component of GA-dependent gene regula-tion despite the fact that the visible growth phenotypeis at least substantially DELLA dependent. Surpris-ingly, regardless of the fact that GA triggers some sim-ilar cellular events during seed germination and floraldevelopment (e.g. GA induces epidermal cell elonga-tion along both the hypocotyl of a germinating seed andthe filament of a growing stamen; Cheng et al., 2004;Cao et al., 2005), the set of genes that are presumablyregulated by DELLAs for seed germination overlaps lit-tle with and is largely distinct from the set of DELLA-regulated genes involved in floral development. Thisobservation suggests that the GA-mediated seed ger-mination and floral development are under the controlof distinct DELLA-dependent transcriptomes.
RESULTS
Identification of DELLA-Dependent Transcriptomesfor Seed Germination
Attempts were made to identify the DELLA-depen-dent transcriptome controlling seed germination byusing oligonucleotide-based DNA microarray analysis(Affymetrix gene chip, carrying 23,000 genes). Seedsof the wild type, ga1-3, and ga1-3 gai-t6 rga-t2 rgl1-1rgl2-1 were imbibed at 4�C for 96 h under continuouswhite light. The cold treatment was included becauseit enhances both the biosynthesis of GA in seeds andthe tissue sensitivity to GA so that it promotes andsynchronizes seed germination (Ogawa et al., 2003;Yamauchi et al., 2004). Total RNA was separatelyextracted from these different seed samples and usedfor microarray analysis to compare their global geneexpression profiles, as described in ‘‘Materials andMethods.’’ Three microarray replicates for each of thethree genotypes in seeds were performed. To minimizethe variation caused by individual hybridization, onlygenes with a logarithm base 2 of the signal ratio of wildtype versus ga1-3 .1 (2-fold higher) or ,21 (2-foldlower) in all three replicates were referred to as GA-up-regulated (GA-up) or GA-down-regulated (GA-down), respectively. Data analysis using the above
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strict criteria identified a total of 541 genes as GA-up(Supplemental Table S1) and 571 genes as GA-down(Supplemental Table S2) in ga1-3 seeds when com-pared to the wild-type control.
We then compared the gene expression patternsbetween ga1-3 and the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1mutant and found that, out of the 541 GA-up genes inga1-3 seeds, mRNA levels of 360 genes (67%) were atleast 2-fold higher in the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1mutant than that in ga1-3 (Table I; Supplemental TableS3), while the remaining 181 genes did not showsignificant changes in their expression (SupplementalTable S4), suggesting that these 360 genes are normallynegatively regulated by DELLAs to repress seed ger-mination. These 360 genes are considered to be DELLAdown-regulated (DELLA-down) and the 181 genes tobe DELLA-independent or -partially-dependent GA-regulated genes. Meanwhile, out of the 571 GA-downgenes in ga1-3 seeds, mRNA levels of 251 genes (44%)were 2-fold lower in the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1mutant than that in ga1-3 (Table I; Supplemental TableS5), while the remaining 320 genes did not showsignificant changes in their expression (SupplementalTable S6), suggesting that these 251 genes are normallypositively regulated by DELLAs to repress seed ger-mination. These 251 genes are considered to be DELLAup-regulated (DELLA-up) and the 320 genes to beDELLA-independent or -partially-dependent GA-regulated genes. To confirm our microarray data,candidate genes were randomly chosen from theDELLA-down and the -up gene list, respectively, andwere subjected to reverse transcription (RT)-PCR anal-ysis using RNA samples independently prepared(Supplemental Table S7). The result showed that all43 genes from the DELLA-down gene list were ex-pressed at higher levels in seeds of both wild type andthe ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant than that inthe ga1-3 seeds, while expression levels of 31/33 genesfrom the DELLA-up gene list were lower in both wildtype and the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutantthan that in ga1-3, exhibiting patterns similar to thatobserved in the microarray analysis (Fig. 1, A and B).The high percentage of confirmation of microarraydata by RT-PCR demonstrates that the microarray datawe obtained are highly reproducible.
Ontology Analysis of DELLA-Dependent Transcriptomes
for Seed Germination
The 360 DELLA-down genes and 251 DELLA-upgenes were subjected to gene ontology analysis usingthe tools and information provided by Affymetrix(NetAffx Gene Ontology Mining Tool), respectively.Among the 360 DELLA-down genes, 257 genes haveeach been assigned a putative molecular functionbased on amino acid homology, and the other 103genes are recorded as functionally unassigned puta-tive genes (NetAffx Gene Ontology Mining Tool; Sup-plemental Table S3). Ontology analysis showed thatthe largest group of DELLA-down genes belongs tothe enzyme genes (total 162 genes, encoding hydro-lase, transferase, and oxidoreductase, etc.) responsiblefor the biosynthesis and metabolism of carbohydrate,protein, nucleotide/nucleic acid, and lipid (Table II), sug-gesting the importance of mobilization of food reservesduring seed germination. The second largest group ofDELLA-down genes contains genes encoding proteinswith binding activity to nucleic acid, nucleotide, ion,and protein binding (total 96 genes; Table II). Furtherexamination of our dataset revealed that seven xylo-glucan endo-1,4-b-D-glucanase genes, five expansingenes, six pectinesterase genes, two endo-1,4-b-glucanasegenes, and one 1,4-b-mannan endohydrolase gene(Table III) were identified as DELLA-down genes. Thesegenes encode well-known factors presumably associ-ated with weakening of the tissue surrounding theembryo to facilitate the embryo growth and radicle pro-trusion (Bewley, 1997; Chen and Bradford, 2000; Chenet al., 2002), suggesting that derepressing DELLAfunction by GA is crucial for the expression of theseimportant cell wall-modifying factors. Interestingly,three a-tubulin genes (TUA2, TUA4, and TUA6) andfour b-tubulin genes (TUB1, TUB5, TUB6, and TUB7)were found as DELLA-down genes (Table III), sug-gesting that DELLA-mediated reorientation of cyto-skeleton might be a key event following cell wallmodification during seed germination (Yuan et al.,1994). In addition, ontology analysis showed that sevenMYB family genes (e.g. MYB4, MYB25, MYB30, MYB34,MYB66), four bHLH family genes (e.g. SPATULA), andfour putative zinc-finger family genes are also foundas DELLA-down genes (Table III). SPATULA (At4g36930)has previously been shown to act as a repressor of seedgermination, probably through repressing the expres-sion of GA 3-oxidase (GA3ox; Penfield et al., 2005).
Among the 251 DELLA-up genes, 150 genes haveeach been assigned a putative molecular function, and101 genes are recorded as expressed putative genes(Supplemental Table S5). As observed for the DELLA-down genes, the two largest groups of DELLA-upgenes are genes encoding enzymes (total 85 genes) andproteins with binding activities (total 79 genes), al-though the total number of DELLA-up enzyme genes(85 genes) is far less than the DELLA-down enzymegenes (162 genes; Table II). Detailed ontology analysisshowed that DELLA-up enzyme genes are mainly for
Table I. Summary of GA- and DELLA-regulated transcriptomes
Criteria used for microarray data analysis are as described in‘‘Materials and Methods.’’ Details are listed in Supplemental TablesS1 (GA-up in seed), S2 (GA-down in seed), S3 (DELLA-down in seed),S5 (DELLA-up in seed), S8 (GA-up in flower bud), S9 (DELLA-down inflower bud), S11 (GA-down in flower bud), and S12 (DELLA-up inflower).
encoding oxidoreductase (30 genes) and transferase(20 genes), while the majority of DELLA-down genesare for hydrolase (70 genes), transferase (50 genes),and oxidoreductase (23 genes; Table II). This resultsuggests that the activity of food metabolism is keptat a low level, while the biosynthetic pathways andenergy production pathways are likely redirected touse a different set of enzymes in the imbibed ga1-3seeds. Surprisingly, a significant number of genesrelated to phytohormonal response (e.g. response toABA, auxin, and ethylene) and stress response/de-fense were identified as DELLA-up genes. These genesinclude 10 ABA-related genes such as responsive todesiccation 29B (RD29B, At5g52300; Uno et al., 2000),ferric iron binding gene ATFER2 (At3g11050; Petit et al.,2001), late embryogenesis abundant M10 (At2g41280;Raynal et al., 1999), etc. Seven auxin-related genes,including AUXIN RESPONSE FACTOR 10 (ARF10;At2g28350; Wang et al., 2005); IAA-LEUCINE RESIS-TANT 1 (ILR1; At3g02875; LeClere et al., 2002); IAA-amido synthase GH3.4 (At1g59500; Staswick et al., 2005);a putative auxin-regulated protein gene (At2g45210);and five ethylene-related genes, including ethylene re-sponsive element binding factor ATERF2 (At5g47220;Fujimoto et al., 2000), ethylene insensitive 3 (EIN3;At5g10120; Riechmann et al., 2000), and ethylene re-sponse factor ERF12 (At1g28360; Ohta et al., 2001),
were also identified (Table III; Supplemental Table S5).In addition, some stress-response genes, including twodehydrin genes (At3g58450, At5g17310) and one su-peroxide dismutase gene (At3g56350) responsive forthe removal of superoxide radicals, are identified asDELLA-up genes (Supplemental Table S5). Comparedwith the relatively large number of DELLA-downgenes in MYB family (seven genes), zinc-finger family(four genes), and bHLH family (four genes), onlytwo zinc-finger family genes (At2g31380, At2g47890)and two bHLH genes (At5g46760, At3g62090) wereidentified as DELLA-up transcription factor genes,whereas none of the MYB family genes was identifiedas DELLA-up genes (Table III).
Identification of DELLA-Dependent Transcriptomes
Expressed during Floral Development
Floral development consists of three distinct phases:floral identity determination (phase transition fromvegetative meristem to an inflorescence meristem),floral organ initiation, and floral organ growth (Krizekand Fletcher, 2005). The development of floral organs,especially petals and stamens, is impaired in GA-deficient mutants, while retarded anthesis results inmale sterility due to a lack of mature pollen (Wilson
Figure 1. RT-PCR confirmation of DELLA-downand DELLA-up genes in the imbibed seeds. A,DELLA-down genes. B, DELLA-up genes. RT-PCRanalysis was repeated on three independent sam-ples and a representative ethidium bromide gelpicture is shown here. Corresponding gene locusidentity (Gene ID) is provided. Two genes(At1g21680 and At3g22490) in B were confirmedin only one of the three repeats but not in othertwo repeats and were marked with an asterisk.Primer pairs for each individual gene are listed inSupplemental Table S7. penta: ga1-3 gai-t6 rga-t2rgl1-1 rgl2-1 penta mutant. ACT2 (ACTIN 2 gene)and UBQ10 (UBIQUITIN 10 gene) were used asthe normalization controls.
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et al., 1992; Cheng et al., 2004). All of the floralphenotypes of the GA-deficient mutant ga1-3 are re-stored to normal in the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1mutant, suggesting that GA signaling through thesefour DELLAs is the major pathway for GA-mediatedfloral development (Cheng et al., 2004; Tyler et al.,2004; Yu et al., 2004). To identify DELLA-dependenttranscriptomes essential for floral development, wealso carried out the microarray assay using RNAsamples extracted from the young and unopenedflower buds of the wild-type control, the ga1-3 mutant,and the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant. Sixmicroarray replicates for each of the three genotypes inseeds were performed. Only genes with a logarithmbase 2 of the signal ratio of wild type versus ga1-3.1 (2-fold higher) or ,21 (2-fold lower) in at leastfour replicates were referred to as GA-up or GA-down,respectively. Based on the above criteria, 826 geneswere identified as GA-up in the ga1-3 young flowerbuds (Supplemental Table S8) when compared to that
in the wild type. The transcript levels of 360 out ofthese 826 GA-up genes (44%) were at least 2-foldhigher in the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutantthan in ga1-3 (Supplemental Table S9), while theremaining 466 genes did not show significant changesin their expression (Supplemental Table S10). These360 genes are supposed to be DELLA-down genes inthe young flower buds, and the 466 genes should beDELLA-independent or -partially-dependent GA-reg-ulated genes. Meanwhile, the transcripts of 422 geneswere accumulated to higher levels in ga1-3 youngflower buds than in the wild type (Supplemental TableS11). The transcript levels of 273 out of these 422 genes(65%) were at least 2-fold lower in the ga1-3 gai-t6 rga-t2rgl1-1 rgl2-1 mutant than in ga1-3 (Supplemental TableS12), while the remaining 149 genes did not showsignificant changes in their expression (Supplemental Ta-ble S13) These 273 genes are supposed to be DELLA-upgenes in the flower buds, and the 149 genes to be DELLA-independent or -partially-dependent GA-regulated
Table II. Ontology analysis of DELLA-regulated genes based on molecular function assigned
In the imbibed seeds, 257/360 DELLA-down (DELLA-D) and 150/251 DELLA-up (DELLA-U) genes wereassigned with putative molecular functions based on amino acid homology. In the young flower buds, 243/360 DELLA-D and 180/273 DELLA-U genes were assigned with molecular functions. Details are providedin Supplemental Tables S3, S5, S9, and S12.
Table III. Cross-comparison of genes related to some important biochemical and biological processes in imbibed seeds and unopened youngflower buds
Genes listed here are summarized from Supplemental Tables S3, S5, S9, and S12 based on information provided in Gene Title, Molecular Function,and Gene Description by Affymetrix. DELLA-D, DELLA-down genes; DELLA-U, DELLA-up genes.
genes. RT-PCR analysis confirmed that all 38 DELLA-down genes and 19 out of 21 DELLA-up genes ran-domly examined exhibited the expected expressionpatterns (Supplemental Table S14; Fig. 2, A and B),demonstrating that the microarray data obtained hereare highly reproducible.
Ontology Analysis of DELLA-Dependent TranscriptomesExpressed during Floral Development
Among the 360 DELLA-repressed genes, 243 geneshave each been assigned a putative molecular functionbased on amino acid homology, and 117 are recordedas functionally unassigned putative genes (Supple-mental Table S9). The majority of DELLA-down floralgenes, as observed for the DELLA-down genes in theimbibed seeds, encode enzymes (total 155 genes) re-sponsible for the metabolism of protein, carbohydrate,and lipid and encode proteins (total 89 genes) withbinding activity to nucleic acid, nucleotide, ion, andprotein binding, suggesting that the arrest of floralorgan growth is coupled with low metabolic activities(Table II). Many types of transcription factors are knownto control or regulate floral development (Krizek andFletcher, 2005). Our microarray analysis identifiedseven MYB family genes, four squamosa promoter-binding protein genes, three bHLH family genes, threeMADS box genes, and three AP2 domain-containingtranscription factor genes as DELLA-down genes(Table III), suggesting these factors might be the linkbetween DELLA-mediated GA signaling and floraldevelopment. Previous studies have shown that theimpaired growth of petal and stamen filament in ga1-3is mainly due to the arrest of cell elongation ratherthan cell division (Cheng et al., 2004). Accordingly, our
microarray analysis showed that DELLAs repress theexpression of genes responsible for the biogenesis andmodification of cell wall components, including fourcellulose synthase genes, four expansin genes, two cel-lulases, and one 1,4-b-mannan endohydrolase (TableIII). GA 2-oxidase (responsible for the degradation ofbioactive GAs) and a GAST1-like gene have previ-ously been shown to be up-regulated by GA (Shi andOlszewski, 1998; Ogawa et al., 2003). The restoration ofexpression of these two genes in ga1-3 gai-t6 rga-t2 rgl1-1rgl2-1 suggests that GA regulates their expressionthrough triggering the degradation of DELLA proteins,and therefore they are identified as DELLA-downgenes (Table III). Interestingly, nine auxin-responsegenes, including auxin-responsive transcription fac-tors IAA19 (At3g15540) and AUXIN RESISTANT 2(AXR2; At3g23050; Liscum and Reed, 2002), putativeIAA-amino acid hydrolase 6 (ILL6; At1g44350; LeClereet al., 2002), two auxin-responsive dopamine beta-monooxygenases (At5g47530 and At3g25290; Neuteboomet al., 1999), and four auxin-responsive genes (At4g13790,At1g29510, At4g12410, and At2g21220; http://www.godatabase.org/cgi-bin/amigo; Table III), are identi-fied as DELLA-down genes in the young flower buds.
Among the 273 DELLA-up floral genes, 180 geneshave each been assigned a putative molecular func-tion, and 93 are recorded as expressed putative genes(Supplemental Table S12). Again, the two largestgroups of DELLA-up genes consist of genes encodingproteins with catalytic activity (total 110 genes) orbinding activity (total 99 genes; Supplemental TableS12). The majority of DELLA-up enzyme genes aretransferase genes (total 55 genes) and oxidoreductasegenes (total 24 genes) but not hydrolase genes, asobserved in DELLA-down floral genes (Table II). GA
biosynthesis is controlled by a negative feedback loop.The lower expression levels of three key GA biosyn-thesis genes (two GA 20-oxidase genes and one GA-3b-hydroxylase gene) in ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1suggest that GA down-regulates these genes throughtriggering the degradation of DELLA proteins, andtherefore they are identified as DELLA-up genes (Ta-ble III; Ogawa et al., 2003). Further analysis showedthat a great range of transcription factors, includingsix putative zinc-finger family genes (ZINC FINGERPROTEIN ZEP3, ZEP4, JAG, etc.; Tague and Goodman,1995; Riechmann et al., 2000), four MYB family genes(MYB59, MYB13, At5g44190, and At3g11280; Riechmannet al., 2000), three putative bHLH family genes(At5g50915, At5g46760, and At4g01460; Heim et al.,2003), and three WRKY family genes (WRKY15,WRKY70, and WRKY53; Eulgem et al., 2000), belongto DELLA-up genes (Table III), suggesting that DELLAsmediate a complex genetic regulation network to re-press floral development. Interestingly, while only twoprotein kinase genes (At5g57670 and At1g61590) andone Leu-rich repeat kinase gene (at4g18640) were iden-tified as DELLA-down genes, a significant number of
putative protein kinase genes (19 protein kinase genes,nine Leu-rich repeat kinase genes, four S-locus proteinkinase genes, and three receptor protein kinase genes)are identified as DELLA-up genes in the young flowerbuds (Table III), suggesting that protein phosphoryla-tion modification might play a key role in controllingfloral organ growth (Morris and Walker, 2003). Sur-prisingly, DELLAs seem to play a crucial active role indefense against disease in the young flower budsbecause 13 disease resistance genes are identified asDELLA-up genes (Table III; Maleck et al., 2000).
DISCUSSION
DELLAs Regulate Distinct Transcriptomes to ControlSeed Germination and Floral Development
Organ initiation, growth, and development are theresult of precisely coordinated action of multiplegenes. The combination of loss-of-function of RGL1,RGL2, RGA, and GAI suppressed the ga1-3 mutantphenotype, and the resultant ga1-3 rgl1-1 rgl2-1 rga-t2gai-t6 mutant confers GA-independent seed germina-tion and floral development, suggesting that thedefective seed germination and floral organ develop-ment in ga1-3 likely result from alteration of the ex-pression of a network of genes that are directly orindirectly regulated by DELLA activity. We are inter-ested to know if a similar set of DELLA-regulatedgenes is used to control these two distinct developmen-tal processes. For this purpose, we compared the geneidentity of the 360 DELLA-down and 251 DELLA-upgenes in the imbibed seeds with that of the 360DELLA-down and 273 DELLA-up genes in the youngflower buds, respectively. Surprisingly, only 21 DELLA-down genes and 15 DELLA-up genes were found to beshared between the two datasets (Supplemental TableS15). RT-PCR analysis confirmed that all 21 sharedDELLA-down genes and 12 out of 14 shared DELLA-up genes examined showed the expected expressionpatterns in the imbibed seed (Fig. 3, A and B). In theyoung flower buds, 18 out of 21 shared DELLA-downgenes and all 13 shared DELLA-up genes examineddisplayed the expected expression patterns (Supple-mental Table S15). Among the 21 DELLA-down genes,only one GAST1-like (At1g74670) gene and two puta-tive expansin genes (At2g37640 and At2g40610) arepresumably related to GA response. Meanwhile, onlyGA-3b-hydroxylase gene (At1g15550) is a known GA-response gene (Ogawa et al., 2003) among the 15DELLA-up genes (Supplemental Table S15). These datademonstrate that GA-mediated seed germination andfloral development are controlled by distinct DELLA-dependent transcriptomes.
Since GA triggers some similar cellular events dur-ing seed germination and floral development (e.g. GAinduces epidermal cell elongation both along thehypocotyl of a germinating seed and the filament ofa growing stamen; Cheng et al., 2004; Cao et al., 2005),the obvious question to ask is how two distinct
Figure 2. RT-PCR confirmation of DELLA-down and DELLA-up genesin the unopened young flower buds. A, DELLA-down genes. B, DELLA-up genes. RT-PCR analysis was repeated on three independent samplesand a representative ethidium bromide gel picture is shown here.Corresponding gene locus identity (Gene ID) is provided. Two genes(At1g09970 and At2g04240) in B did not show obvious difference inexpression and were marked with an asterisk. Primer pairs for eachindividual gene are listed in Supplemental Table S12. penta: ga1-3 gai-t6rga-t2 rgl1-1 rgl2-1 penta mutant. ACT2 (ACTIN 2 gene) was used as thenormalization control.
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DELLA-dependent transcriptomes regulate similar cel-lular events. To address this question, we first sub-grouped the DELLA-regulated genes, identified eitherin the imbibed seeds or young flower buds, based ontheir known or presumable molecular functions inplanta. Next, we cross-compared genes in correspond-ing subgroups in the dataset for imbibed seeds anddataset for young flower buds.
Novel GAMYB Genes and Other Transcription Factors
DELLAs are putative transcription regulators. Pre-sumably, they may directly regulate the expression ofsome GA-response genes. Unfortunately, there is cur-rently no concrete evidence to prove this hypothesis.Alternatively, DELLAs regulate the expression of
some downstream transcription factors, and theseDELLA-regulated transcription factors then controlthe expression of GA-response genes. GAMYB genesare the best studied GA-regulated transcription fac-tors, and previous studies have shown that GA regu-lates GAMYB through DELLA proteins SLN and SLRin barley (Hordeum vulgare) and rice (Oryza sativa),respectively (Gubler et al., 2002; Kaneko et al., 2003). InArabidopsis, MYB33 and MYB65 are identified asGAMYB genes based on homology analysis. However,the MYB33 and MYB65 and their subfamily members areregulated at the posttranscription level by miRNA159(Achard et al., 2004; Millar and Gubler, 2005). In fact,MYB33 and MYB65 are not identified among theDELLA-down or DELLA-up genes in our dataset.On the other hand, our data showed that MYB4,MYB25, MYB30, MYB34, MYB66, and two MYB homo-logs (At1g01380 and At5g58900) are the seven DELLA-down MYB genes involved in seed germination, whilethe other seven DELLA-down MYB genes (MYB24,MYB32, MYB52, MYB106, MYB21, MYB, and At2g38090)are involved in floral development (Table III), suggest-ing that DELLAs differentially regulate a different sub-set of MYB genes to repress seed germination andfloral development. Interestingly, four MYBs (MYB59,MYB At5g44190, MYB At1g06180, and MYB At3g11280)were identified as DELLA-up genes in the youngflower buds, while no DELLA-up MYB gene wasfound in the imbibed seeds (Table III). Therefore, theseMYB genes may represent new types of GAMYBs, andfuture work will focus on studying the relationshipbetween GA and these MYB genes. In addition to MYBgenes, distinct DELLA-down or -up bHLH and zinc-finger family genes are also identified both in theimbibed seeds and young flower buds (Table III). It isinteresting to note that four zinc-finger family geneswere identified as DELLA-down genes in the imbibedseeds, while six other zinc-finger family genes wereidentified as DELLA-up genes in the young flowerbuds, indicating that the zinc-finger gene family isdifferentially regulated by DELLAs at different devel-opmental stages. As expected, three types of transcrip-tion factors, namely, three MADS box family genes(AGL1, AGL6, and AGL11), three WRKY family genes(WRKY15, WRKY70, and WRKY53; Eulgem et al., 2000),and five squamosa promoter-binding protein-box familygenes (SPL2, SPL5, SPL11, and SPL12), are found amongthe DELLA-regulated genes only for floral develop-ment (Table III; Krizek and Fletcher, 2005). Apparently,these transcription factors will target their own spe-cific targets to fine-tuning the regulation initiated byDELLAs. One of the future tasks will be to find out thetargets controlled by these transcription factors.
Protein Phosphorylation Might Represent a MajorPathway for DELLA Repression of FloralOrgan Development
Protein phosphorylation and dephosphorylation iswidely involved in signaling cascade to trigger the
Figure 3. RT-PCR confirmation of shared DELLA-down and DELLA-upgenes in the imbibed seeds and young flower buds. A, Shared-DELLA-down genes. B, Shared-DELLA-up genes. RT-PCR analysis was repeatedon three independent samples and a representative ethidium bromidegel picture is shown here. Corresponding gene locus identity (Gene ID)is provided. Three shared DELLA-down genes and two shared DELLA-up genes showed no difference in expression in the young flower budsand imbibed seeds, respectively, and these genes were marked with anasterisk. Primer pairs for each individual gene are listed in Supplemen-tal Table S13. penta: ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 penta mutant. Forthe imbibed seeds, ACT2 (ACTIN 2 gene) and UBQ10 (UBIQUITIN 10gene) were used as the normalization controls. For the young flowerbuds, ACT2 was used as the normalization control.
DELLA-Dependent Transcriptomes in Seed and Flower
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downstream cellular events. Six DELLA-down andnine DELLA-up protein kinase genes are identified inthe imbibed seeds (Table III), suggesting that thechange of phosphorylation status of signaling proteinsis probably involved in causing the nongerminatingphenotype of the ga1-3 mutant. Surprisingly, micro-array analysis revealed that DELLAs regulate theprotein kinase families in a unique way during floraldevelopment. About 21 DELLA-up protein kinasegenes were identified in the young flower buds, whileonly two were found as DELLA-down genes (TableIII). In addition, while the expression of nine Leu-richrepeat protein kinase genes was repressed by DELLAsin the imbibed seeds, a completely different set of nineLeu-rich repeat protein kinase genes was up-regulatedby DELLAs in the young flower buds (Table III).Furthermore, five S-locus-related protein kinase geneswere activated by DELLAs in the young flower budsonly (Table III). Therefore, DELLAs differentially reg-ulate the expression of different protein kinase genesto control seed germination and floral development.Combining all data, it seems that activating proteinphosphorylation pathways might be a crucial step forDELLAs to repress floral development.
DELLAs Maintain the Low Metabolic Activity in thega1-3 Mutant
Seed germination is an active process that needs tomobilize food reserves to provide sufficient energyand building blocks to sustain the dynamic cellularactivities in the germinating seed. In contrast, a non-germinating seed normally maintains low metabolicactivity (Bewley, 1997). In both imbibed seeds andyoung flower buds, a large number of genes encodingenzymes (especially hydrolase, transferase, and oxido-reductase) responsible for the metabolism of carbon-hydrate, protein, and lipid are repressed by DELLAs(Table II; Supplemental Tables S5, S9, and S14). Thisfact suggests that the metabolic activities in bothimbibed ga1-3 mutant seeds and young ga1-3 mutantflower buds are likely kept at a low level, and this lowmetabolic activity of mobilization of food reservesnicely correlates with the nongerminating and arres-ted floral development phenotypes displayed by thega1-3 mutant. When compared to the wild type, thetranscript levels of a large number of different trans-ferase and oxidoreductase genes in ga1-3 were alteredin the imbibed seeds and young flower buds, respec-tively (Table II; Supplemental Table S16), suggestingthat biosynthetic and oxidative pathways are redirec-ted to other pathways in the ga1-3 mutant. Cross-comparison showed that the identities (gene locus) ofthe DELLA-regulated (both -repressed and -activated)hydrolase genes, transferase genes, oxidoreductase,and other enzyme genes in the imbibed seeds are al-most completely different from their respective coun-terparts in DELLA-regulated enzyme genes in the youngflower buds (Table II; Supplemental Table S16). Thisfact strongly suggests that DELLAs differentially reg-
ulate different subsets of metabolic genes of similarmolecular functions or different individual membersof a same gene family to control seed germination andfloral development.
Distinct Approaches Are Utilized to Control Cell
Growth and Cell Wall Modification during SeedGermination and Floral Development
Prior to seed germination, a number of cell wall-modifying genes will be activated to loosen the cellwall and break the seed coat to facilitate the radicleprotrusion. Similarly, during the period of the floralorgan growth, factors will be produced to promote theelongation of epidermal cells of petal, stamen, andpistil. Five and four expansin genes were identified asDELLA-down genes in the imbibed seeds and youngflower buds, respectively, and two of them (At2g37640and At2g40610) are shared (Table III), suggesting thatexpansins are crucial for the cell elongation in bothdevelopmental processes. However, while seven xy-loglucan endotransglycosylase/hydrolase and six pec-tinesterase genes are the major genes responsible forthe cell wall loosening in the imbibed seeds, none ofthese two categories of genes was DELLA-down in theyoung flower buds (Table III). Instead, four cellulosesynthase genes were found as DELLA-down genesonly in the young flower buds but not in the imbibedseeds (Table III; Supplemental Tables S3 and S9).Interestingly, three a-tubulin genes (TUA2, TUA4,and TUA6) and four b-tubulin genes (TUB1, TUB5,TUB6, and TUB7) are also DELLA-down genes in theimbibed seeds but not in the young flower buds (TableIII). These results suggest that the cell elongationactivity during seed germination is probably mainlyresulted from cell wall loosening coupled with cellreshaping, while the cell elongation during floraldevelopment is mainly due to the de novo biosynthe-sis of cellulose.
DELLAs Act as Convergence Point for
Phytohormone Signaling
As expected, in both imbibed seeds and youngflower buds, the GA-response gene GAST1(At1g74670) and the key GA biosynthesis gene GA-3-b-hydroxylase (At1g15550) are identified as DELLA-down and -up genes, respectively (Shi and Olszewski,1998; Ogawa et al., 2003). Recently, Ueguchi-Tanaka(Ueguchi-Tanaka et al., 2005) reported that OsGID1 inrice encodes a soluble GA-receptor with homology tothe consensus sequence of the hormone-sensitive li-pase (HSL) homologous family. A database searchidentified three OsGID1 homologs in Arabidopsis (Fig.4), and all of them have recently been shown to bindGA (Nakajima et al., 2006). Interestingly, two of theseOsGID1 homologs (At3g05120 and At3g63010) areidentified as DELLA-up genes in the young flowerbuds and one (At3g05120) in the imbibed seeds, sug-gesting that these OsGID1 homologs are probably
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negatively regulated by GA. However, the fact thatDELLA proteins are stabilized in the ga1-3 mutantsuggests that GA is necessary to activate the GID1-likereceptors to trigger the degradation of DELLA pro-teins. A total of 14 GDSL-type lipase genes, anothertype of lipase presumably related to defense (Akohet al., 2004), are identified as DELLA-down genes(Table IV; Supplemental Fig. S1). Previous studiesshowed that ABA signaling through ABI1 and ethyl-ene signaling through CTR1 enhance the stability ofDELLAs (Achard et al., 2003, 2006), implying that afraction of ABA- and ethylene-signaling responsegenes will probably be identified as DELLA-regulatedgenes in our dataset. Indeed, a number of ABA- andethylene-response genes were identified as DELLA-upgenes in both the imbibed seeds and young flowerbuds. For example, the expression of late-embryogen-esis-abundant (LEA) proteins genes in the imbibedseeds is known to be responsive to ABA treatment(Ali-Benali et al., 2005; Kamisugi and Cuming, 2005;Bethke et al., 2006), and seven of these LEA proteingenes were identified as DELLA-up genes (Table III).Also, five and three ethylene-related genes were foundas DELLA-up genes in the imbibed seeds and youngflower buds, respectively. These genes include genesfor ethylene responsive element binding factor1 (ERF2; At5g47220; McGrath et al., 2005), EIN3,ethylene responsive element binding factor (ERF12;At1g28360), ethylene-responsive element-bindingfamily protein (At5g61600), and universal stress pro-
tein USP/ER6 (At3g58450; Table III; SupplementalTables S5 and S12; Chang and Bleecker, 2004; Guoand Ecker, 2004). That low concentrations of auxin pro-mote the destabilization of DELLAs (Fu and Harberd,2003) fits well with the finding that four (the auxinefflux carrier EIR1, auxin-induced protein AIR12, AIR9,and At4g34760; Luschnig et al., 1998; Neuteboom et al.,1999) and nine auxin-response genes were identifiedas DELLA-down genes in the imbibed seeds and youngflower buds, respectively. Interestingly, seven auxin-related genes, including genes encoding IAA-aminoacid hydrolase ILR1 (At3g02875), auxin-regulated pro-tein GH3 (At1g59500), auxin-induced protein IAA17/AXR3-1, and two auxin-induced proteins similar toauxin-induced atb2 (At1g60710 and At1g60680; TableIII; Supplemental Table S5; Liscum and Reed, 2002),were identified as DELLA-up genes in the imbibedseeds, suggesting that the interaction between GA andauxin is probably more complicated than previouslythought.
In Addition to Protecting Plant from AdverseEnvironment, DELLAs Might Also MediateDisease Resistance in Young Flower Buds
Recent studies have shown that DELLAs act as theintegrator of environmental cues and endogenousphytohormonal signals to protect plants from theenvironmental stress (Lee et al., 2002; Achard et al.,2003, 2006; Cao et al., 2005). For both the imbibed seeds
Figure 4. Amino acid sequence alignment of rice GID1 with its three Arabidopsis homologs (At3g05120, At3g63010, andAt5g27320) using the ClustalW program. Gene ID is provided on the left side of the figure.
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and young flower buds, genes responsive to oxidativestress were identified as DELLA-down genes (TableIII). On the other hand, genes responsive to waterstress (dehydration/desiccation) and genes responsi-ble for toxin catabolism are more sensitive to DELLAregulation in the imbibed seeds than in the youngflower buds. Interestingly, six water stress responsegenes were identified as DELLA-down genes and13 water stress response genes were identifiedas DELLA-up genes in the imbibed seeds (Table III),suggesting there might be a switch of water stressresponse pathways during different stages of plantgrowth. In contrast, multidrug transport and wound-ing response genes were identified as DELLA-downgenes mainly in the young flower buds (Table III).Most significantly, a great number of putative diseasedefense genes (13 genes) were identified as DELLA-upgenes (Table III), implying that DELLAs are not onlyactively involved in protecting plant from differentenvironmental stress but probably in mediating dis-ease resistance as well, especially during stages ofplant growth.
DELLA-Independent or -Partially-Dependent
GA-Regulated Genes
Gene expression profiling data in Arabidopsis andrice has shown that a wide range of genes, includinggenes encoding enzymes and other factors that de-grade the cell wall of endosperm and seed coat, areregulated by GA to stimulate the growth of the em-bryo, elongation of the embryo axis, and breakage ofseed coat (Ogawa et al., 2003; Bethke et al., 2006).Ogawa et al. compared the expression profiles be-tween GA-treated and untreated ga1-3 seeds at varioustime points after 48-h stratification in the dark and 24 hat 22�C in the light and identified a total of 230 GA-upgenes and 127 GA-down genes using an Arabidopsisgene chip carrying approximately 8,200 genes (Ogawaet al., 2003). We cross-compared the 541 GA-up genesand 571 GA-down genes obtained in our experimentwith the 230 GA-up genes and 127 GA-down genesidentified by Ogawa et al., respectively, and found that109 GA-up genes (approximately 47% of 230 genesobtained by Ogawa et al.) and 90 GA-down genes(approximately 71% of 127 genes obtained by Ogawaet al.) are shared in both datasets (Supplemental Tables
S17 and S18). Given the differences in the experimentaldesign, the high degree of overlap between the twodatasets is quite impressive. More interestingly, fur-ther analysis showed that 91 out of the 109 shared GA-up genes were among the 360 DELLA-down genes (andthus are regulated in a DELLA-dependent fashion) inthe imbibed seeds, while the remaining 18 sharedGA-up genes were among the 181 DELLA-independentor -partially-dependent genes (Supplemental TableS17). Meanwhile, data analysis also showed that 56out of 90 shared GA-down genes were among the251 DELLA-up genes (and thus are DELLA dependent)in the imbibed seeds. However, the remaining 34shared GA-down genes were among the 320 DELLA-independent or -partially-dependent genes (Supple-mental Table S18). Because the GA-regulated genes inOgawa’s dataset were obtained by applying GA to thega1-3 seeds, the above data support the hypothesis thatthere is probably an unknown DELLA-independent or-partially-dependent component essential for the regu-lation of some GA-dependent genes.
CONCLUSION
In this report, we identified GA-regulated (both GA-down and -up) transcriptomes in both imbibed seedsand young flower buds by comparing the expressionpatterns between the ga1-3 mutant and wild-type con-trol. Then, we identified DELLA-dependent (bothDELLA-down and -up) transcriptomes by finding outthe subgroup of GA-regulated genes with their expres-sion restored to the wild-type levels in the ga1-3 rga-t2gai-t6 rgl1-1 rgl2-1 mutant. The high percentage ofoverlap between GA-regulated genes identified in ourwork and Ogawa’s work, together with the high rate ofconfirmation of candidate genes by RT-PCR analysis,demonstrate that the datasets obtained are highly re-producible and reliable. The complete suppression ofga1-3 nongerminating and male sterile phenotypes byloss-of-function of RGA, GAI, RGL1, and RGL2 impliesthat GA-dependent gene regulation might be largelythrough the DELLA-dependent pathway. Interestingly,we observed that approximately half of total GA-regulated genes are regulated via the DELLA-dependentpathway, suggesting an unknown DELLA-independentcomponent is probably essential for the regulation of
Table IV. GDSL-type lipase genes regulated by DELLAs
other GA-dependent genes. However, because wehave set strict criteria to identify the DELLA-regulatedgenes, we might have missed identifying some DELLA-regulated genes due to sample variations and alsocannot exclude the possibility that a portion of theremaining GA-regulated genes might be partially reg-ulated by DELLAs. Finally, we cross-compared theDELLA-dependent transcriptomes between imbibedseeds and young flower buds and surprisingly foundthat, based on gene identity (gene locus ID), the twoDELLA-dependent transcriptomes are almost entirelydistinct from each other. Ontology analysis revealedthat a large number of genes with similar molecularand biochemical functions (e.g. genes for hydrolases,transferases, oxidoreductases, proteins with bindingactivity, MYBs, bHLHs, expansins, etc.) are repressedor up-regulated by DELLAs in both imbibed seeds andyoung flower buds. In fact, these groups of genesconstitute the largest portion of DELLA-dependenttranscriptomes in both imbibed seeds and youngflower buds. This fact suggests that the many basicbiochemical pathways are similarly mobilized duringseed germination and floral development. However,specific factors participating in these pathways aredifferent individual members from different gene fam-ilies, suggesting that DELLAs differentially regulatesthe expression of these specific factors during seedgermination and floral development. Meanwhile, de-tailed data analysis revealed that DELLAs also controlthe expression of many functionally completely differ-ent genes, including factors for cell wall loosening,stress and disease response, and protein phosphoryl-ation modification, to run different pathways eitherspecific for seed germination or for floral development,which signifies the differences between these two im-portant biological processes. In conclusion, the datashown here not only confirm the results obtained frommany previous reports but also single out some novelaspects of DELLA functions that will be instructive toour future research.
