A Novel 10-Base Pair Deletion in the First Exon of GmHY2a Promotes Hypocotyl Elongation, Induces Early Maturation, and Impairs Photosynthetic Performance in Soybean.

Plants photoreceptors perceive changes in light quality and intensity and thereby regulate plant vegetative growth and reproductive development. By screening a γ irradiation-induced mutant library of the soybean (Glycine max) cultivar "Dongsheng 7", we identified Gmeny, a mutant with elongated nodes, yellowed leaves, decreased chlorophyll contents, altered photosynthetic performance, and early maturation. An analysis of bulked DNA and RNA data sampled from a population segregating for Gmeny, using the BVF-IGV pipeline established in our laboratory, identified a 10 bp deletion in the first exon of the candidate gene Glyma.02G304700. The causative mutation was verified by a variation analysis of over 500 genes in the candidate gene region and an association analysis, performed using two populations segregating for Gmeny. Glyma.02G304700 (GmHY2a) is a homolog of AtHY2a in Arabidopsis thaliana, which encodes a PΦB synthase involved in the biosynthesis of phytochrome. A transcriptome analysis of Gmeny using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed changes in multiple functional pathways, including photosynthesis, gibberellic acid (GA) signaling, and flowering time, which may explain the observed mutant phenotypes. Further studies on the function of GmHY2a and its homologs will help us to understand its profound regulatory effects on photosynthesis, photomorphogenesis, and flowering time.

A rice seed-specific glycine-rich protein OsDOR1 interacts with GID1 to repress GA signaling and regulates seed dormancy.

Seed dormancy is an important agronomic trait under the control of complex genetic and environmental interactions, which have not been yet comprehensively understood. From the field screening of rice mutant library generated by a Ds transposable element, we identified a pre-harvest sprouting (PHS) mutant dor1. This mutant has a single insertion of Ds element at the second exon of OsDOR1 (LOC_Os03g20770), which encodes a novel seed-specific glycine-rich protein. This gene successfully complemented the PHS phenotype of dor1 mutant and its ectopic expression enhanced seed dormancy. Here, we demonstrated that OsDOR1 protein binds to the GA receptor protein, OsGID1 in rice protoplasts, and interrupts with the formation OsGID1-OsSLR1 complex in yeast cells. Co-expression of OsDOR1 with OsGID1 in rice protoplasts attenuated the GA-dependent degradation of OsSLR1, the key repressor of GA signaling. We showed the endogenous OsSLR1 protein level in the dor1 mutant seeds is significantly lower than that of wild type. The dor1 mutant featured a hypersensitive GA-response of α-amylase gene expression during seed germination. Based on these findings, we suggest that OsDOR1 is a novel negative player of GA signaling operated in the maintenance of seed dormancy. Our findings provide a novel source of PHS resistance.

Analysis of morphological traits and regulatory mechanism of a semi-dwarf, albino, and blue grain wheat line.

The plant height and leaf color are important traits in crops since they contribute to the production of grains and biomass. Progress has been made in mapping the genes that regulate plant height and leaf color in wheat (Triticum aestivum L.) and other crops. Wheat line DW-B (dwarfing, white leaves, and blue grains) with semi-dwarfing and albinism at the tillering stage and re-greening at the jointing stage was created using Lango and Indian Blue Grain. Transcriptomic analyses of the three wheat lines at the early jointing stages indicated that the genes of gibberellin (GA) signaling pathway and chlorophyll (Chl) biosynthesis were expressed differently in DW-B and its parents. Furthermore, the response to GA and Chl contents differed between DW-B and its parents. The dwarfing and albinism in DW-B were owing to defects in the GA signaling pathway and abnormal chloroplast development. This study can improve understanding of the regulation of plant height and leaf color. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01379-z.

PROTEIN PHOSPHATASE 2C08, a Negative Regulator of Abscisic Acid Signaling, Promotes Internode Elongation in Rice.

Clade A protein phosphatase 2Cs (PP2CAs) negatively regulate abscisic acid (ABA) signaling. Here, we investigated the functions of OsPP2CAs and their crosstalk with ABA and gibberellic acid (GA) signaling pathways in rice (Oryza sativa). Among the nine OsPP2CAs, OsPP2C08 had the highest amino acid sequence similarity with OsPP2C51, which positively regulates GA signaling in rice seed germination. However, OsPP2C08 was expressed in different tissues (internodes, sheaths, and flowers) compared to OsPP2C51, which was specifically expressed in seeds, and showed much stronger induction under abiotic stress than OsPP2C51. Transgenic rice lines overexpressing OsPP2C08 (OsPP2C08-OX) had a typical ABA-insensitive phenotype in a post-germination assay, indicating that OsPP2C08, as with other OsPP2CAs, negatively regulates ABA signaling. Furthermore, OsPP2C08-OX lines had longer stems than wild-type (WT) plants due to longer internodes, especially between the second and third nodes. Internode cells were also longer in OsPP2C08-OX lines than in the WT. As GA positively regulates plant growth, these results suggest that OsPP2C08 might positively regulate GA biosynthesis. Indeed, the expression levels of GA biosynthetic genes including gibberellin 20-oxidase (OsGA20ox4) and Ent-kaurenoic acid oxidase (OsKAO) were increased in OsPP2C08-OX lines, and we observed that GIBBERELLIN 2-OXIDASE 4 (OsGA2ox4), encoding an oxidase that catalyzes the 2-beta-hydroxylation of several biologically active GAs, was repressed in the OsPP2C08-OX lines based on a transcriptome deep sequencing and RT-qPCR analysis. Furthermore, we compared the accumulation of SLENDER RICE 1 (SLR1), a DELLA protein involved in GA signaling, in OsPP2C08-OX and WT plants, and observed lower levels of SLR1 in the OsPP2C08-OX lines than in the WT. Taken together, our results reveal that OsPP2C08 negatively regulates ABA signaling and positively regulates GA signaling in rice. Our study provides valuable insight into the molecular mechanisms underlying the crosstalk between GA and ABA signaling in rice.

Genome-Wide Analysis of Genes Involved in the GA Signal Transduction Pathway in 'duli' Pear (Pyrus betulifolia Bunge).

Gibberellic acid (GA) is an important phytohormone that regulates every aspect of plant growth and development. While elements involved in GA signaling have been identified and, hence, their functions have been well studied in model plants, such as Arabidopsis and rice, very little is known in pear. We, therefore, analyzed the genes related to GA signaling from the recently sequenced genome of the wildtype 'duli' pear (Pyrus betulifolia Bunge), a widely used rootstock for grafting in pear cultivation in China due to its vigorous growth and resistance to abiotic and biotic stress. In total, 15 genes were identified, including five GA receptors PbGID1s (GA-INSENSTIVE DWARF 1), six GA negative regulators, PbDELLAs, and four GA positive regulators, PbSLYs. Exogenous application of GA could promote the expression of PbGID1s but inhibit that of PbDELLAs and PbSLYs in tissue culture 'duli' pear seedlings. The expression profiles of these genes in field-grown trees under normal growth conditions, as well as in tissue-cultured seedlings treated with auxin (IAA), GA, paclobutrazol (PAC), abscisic acid (ABA), and sodium chloride (NaCl), were also studied, providing further evidence of the involvement of these genes in GA signaling in 'duli' pear plants. The preliminary results obtained in this report lay a good foundation for future research into GA signaling pathways in pear. Importantly, the identification and preliminary functional verification of these genes could guide molecular breeding in order to obtain the highly desired dwarf pear rootstocks for high-density plantation to aid easy orchard management and high yielding of pear fruits.

Identification of BcARR Genes and CTK Effects on Stalk Development of Flowering Chinese Cabbage.

Flowering Chinese cabbage (Brassica campestris L. ssp. Chinensis var. utilis Tsen et Lee) is an important and extensively cultivated vegetable in south China, whose major food product is the stalk. In the process of stalk formation, its initiation and development are regulated by a series of hormonal signals, such as cytokinin and gibberellin. In this study, we analyzed the effects of zeatin (ZT) and gibberellin A3 (GA3), and their interaction, on the bolting of flowering Chinese cabbage. The results indicated that the three-true-leaf spraying of ZT and GA synthesis inhibitor (PAC) inhibited plant height but increased stem diameter. Cytokinin (CTK) synthesis inhibitor (YZJ) and GA3 treatment increased plant height and decreased stem diameter. In addition, ZT and GA3 co-treated plants displayed antagonistic effect. Further, 19 type-B authentic response regulators (ARR-Bs), the positive regulators of cytokinin signal transduction were identified from flowering Chinese cabbage. Comprehensive analysis of phylogeny showed BcARR-Bs clustered into three subfamilies with 10 conserved motifs. Analysis of their expression patterns in different tissues and at various growth stage, and their response to hormone treatment suggest that ARR1-b localized in the nucleus displayed unique highest expression patterns in stem tips, are responsive both to ZT and GA, suggesting a significant role in mediating the crosstalk of ZT and GA in the bolting of flowering Chinese cabbage.

Functional characterization of a gibberellin F-box protein, PslSLY1, during plum fruit development.

Fruit development is orchestrated by a complex network of interactions between hormone signaling pathways. The phytohormone gibberellin (GA) is known to regulate a diverse range of developmental processes; however, the mechanisms of GA action in perennial fruit species are yet to be elucidated. In the current study, a GA signaling gene PslSLY1, encoding a putative F-box protein that belongs to the SLY1 (SLEEPY1)/GID2 (gibberellin-insensitive dwarf2) gene family, was isolated from Japanese plum (Prunus salicina). PslSLY1 transcript abundance declined as fruit development progressed, along with potential negative feedback regulation of PslSLY1 by GA. Subcellular localization and protein-protein interaction assays suggested that PslSLY1 functions as an active GA signaling component that interacts with the ASK1 (Arabidopsis SKP1) subunit of an SCF-ubiquitin ligase complex and with PslDELLA repressors, in a GA-independent manner. By using a domain omission strategy, we illustrated that the F-box and C-terminal domains of PslSLY1 are essential for its interactions with the downstream GA signaling components. PslSLY1 overexpression in wild-type and Arabidopsissly1.10 mutant backgrounds resulted in a dramatic enhancement in overall plant growth, presumably due to triggered GA signaling. This includes germination characteristics, stem elongation, flower structure, and fertility. Overall, our findings shed new light on the GA strategy and signaling network in commercially important perennial crops.

Arabidopsis G-Protein ß Subunit AGB1 Negatively Regulates DNA Binding of MYB62, a Suppressor in the Gibberellin Pathway.

Plant G proteins are versatile components of transmembrane signaling transduction pathways. The deficient mutant of heterotrimeric G protein leads to defects in plant growth and development, suggesting that it regulates the GA pathway in Arabidopsis. However, the molecular mechanism of G protein regulation of the GA pathway is not understood in plants. In this study, two G protein ß subunit (AGB1) mutants, agb1-2 and N692967, were dwarfed after exogenous application of GA3. AGB1 interacts with the DNA-binding domain MYB62, a GA pathway suppressor. Transgenic plants were obtained through overexpression of MYB62 in two backgrounds including the wild-type (MYB62/WT Col-0) and agb1 mutants (MYB62/agb1) in Arabidopsis. Genetic analysis showed that under GA3 treatment, the height of the transgenic plants MYB62/WT and MYB62/agb1 was lower than that of WT. The height of MYB62/agb1 plants was closer to MYB62/WT plants and higher than that of mutants agb1-2 and N692967, suggesting that MYB62 is downstream of AGB1 in the GA pathway. qRT-PCR and competitive DNA binding assays indicated that MYB62 can bind MYB elements in the promoter of GA2ox7, a GA degradation gene, to activate GA2ox7 transcription. AGB1 affected binding of MYB62 on the promoter of GA2ox7, thereby negatively regulating th eactivity of MYB62.

Small grain and semi-dwarf 3, a WRKY transcription factor, negatively regulates plant height and grain size by stabilizing SLR1 expression in rice.

KEY MESSAGE: OsWRKY36 represses plant height and grain size by inhibiting gibberellin signaling. Plant height and grain size are important agronomic traits affecting yield in cereals, including rice. Gibberellins (GAs) are plant hormones that promote plant growth and developmental processions such as stem elongation and grain size. WRKYs are transcription factors that regulate stress tolerance and plant development including height and grain size. However, the relationship between GA signaling and WRKY genes is still poorly understood. Here, we characterized a small grain and semi-dwarf 3 (sgsd3) mutant in rice cv. Hwayoung (WT). A T-DNA insertion in the 5'-UTR of OsWRKY36 induced overexpression of OsWRKY36 in the sgsd3 mutant, likely leading to the mutant phenotype. This was confirmed by the finding that overexpression of OsWRKY36 caused a similar small grain and semi-dwarf phenotype to the sgsd3 mutant whereas knock down and knock out caused larger grain phenotypes. The sgsd3 mutant was also hyposensitive to GA and accumulated higher mRNA and protein levels of SLR1 (a GA signaling DELLA-like inhibitor) compared with the WT. Further assays showed that OsWRKY36 enhanced SLR1 transcription by directly binding to its promoter. In addition, we found that OsWRKY36 can protect SLR1 from GA-mediated degradation. We thus identified a new GA signaling repressor OsWRKY36 that represses GA signaling through stabilizing the expression of SLR1.

Interplay between REVEILLE1 and RGA-LIKE2 regulates seed dormancy and germination in Arabidopsis.

Environmental light signal and GAs synergistically regulate seed dormancy and germination. The phytochrome B (phyB) photoreceptor regulates expression of the REVEILLE1 (RVE1) transcription factor, which directly inhibits GIBBERELLIN 3-OXIDASE2 transcription, suppressing GA biosynthesis. However, whether phyB-RVE1 coordinates with GA signaling in controlling seed dormancy and germination remains unknown. Here, we demonstrate that RVE1 regulation of seed dormancy and germination requires a DELLA repressor, REPRESSOR OF GA-LIKE2 (RGL2), in Arabidopsis thaliana. RVE1 interacts with both RGL2 and its E3 ubiquitin ligase SLEEPY1 (SLY1) and promotes RGL2 stability by restraining the RGL2-SLY1 interaction. Furthermore, RVE1 and RGL2 synergistically regulate global transcriptome changes; RGL2 enhances the DNA-binding capacity and transcriptional activity of RVE1 in regulating downstream gene expression. Moreover, RGL2 expression is repressed by phyB. Our study reveals a novel regulatory mechanism in which the RVE1-RGL2 module coordinately controls seed dormancy and germination by integrating light perception, GA metabolism and GA signaling pathways.

NAP is involved in GA-mediated chlorophyll degradation and leaf senescence by interacting with DELLAs in Arabidopsis.

KEY MESSAGE: RGA/GAI and NAP interacted with each other, and NAP was involved in GA signaling as a role of regulating age-dependent and dark-induced leaf senescence in Arabidopsis. Leaf senescence is a significant biological process which is beneficial for plant growth, development, and generation alternation in Arabidopsis. Recent researches have shown gibberellins (GAs) could accelerate leaf senescence. Nevertheless, the GA signaling involved in leaf senescence process remains elusive. Here, we reported a new potential regulation mechanism of GA-mediated chlorophyll degradation and leaf senescence. In this study, we confirmed that NAP positively regulated age-dependent and dark-induced leaf senescence and NAP knockout mutant nap was hyposensitive to GA3 (an active form of GA) treatment. DELLA family proteins with highly conserved structural domain function as master growth repressors that integrated GA signaling and leaf senescence. We validated RGA and GAI could interact with NAP in vitro and in vivo, and subsequently impaired the transcriptional activities of NAP to induce SAG113 and AAO3 expression in nap protoplasts. Taken together, we suggest that NAP is a novel component of the regulatory network that modulates the progress of leaf senescence in GA signaling.

Generation and Transcriptome Profiling of Slr1-d7 and Slr1-d8 Mutant Lines with a New Semi-Dominant Dwarf Allele of SLR1 Using the CRISPR/Cas9 System in Rice.

The rice SLR1 gene encodes the DELLA protein, and a loss-of-function mutation is dwarfed by inhibiting plant growth. We generate slr1-d mutants with a semi-dominant dwarf phenotype to target mutations of the DELLA/TVHYNP domain using CRISPR/Cas9 genome editing in rice. Sixteen genetic edited lines out of 31 transgenic plants were generated. Deep sequencing results showed that the mutants had six different mutation types at the target site of the TVHYNP domain of the SLR1 gene. The homo-edited plants selected individuals without DNA (T-DNA) transcribed by segregation in the T1 generation. The slr1-d7 and slr1-d8 plants caused a gibberellin (GA)-insensitive dwarf phenotype with shrunken leaves and shortened internodes. A genome-wide gene expression analysis by RNA-seq indicated that the expression levels of two GA-related genes, GA20OX2 (Gibberellin oxidase) and GA3OX2, were increased in the edited mutant plants, suggesting that GA20OX2 acts as a convert of GA12 signaling. These mutant plants are required by altering GA responses, at least partially by a defect in the phytohormone signaling system process and prevented cell elongation. The new mutants, namely, the slr1-d7 and slr1-d8 lines, are valuable semi-dominant dwarf alleles with potential application value for molecule breeding using the CRISPR/Cas9 system in rice.

NO FLOWERING IN SHORT DAY (NFL) is a bHLH transcription factor that promotes flowering specifically under short-day conditions in Arabidopsis.

Flowering in plants is a dynamic and synchronized process where various cues including age, day length, temperature and endogenous hormones fine-tune the timing of flowering for reproductive success. Arabidopsis thaliana is a facultative long day (LD) plant where LD photoperiod promotes flowering. Arabidopsis still flowers under short-day (SD) conditions, albeit much later than in LD conditions. Although factors regulating the inductive LD pathway have been extensively investigated, the non-inductive SD pathway is much less understood. Here, we identified a key basic helix-loop-helix transcription factor called NFL (NO FLOWERING IN SHORT DAY) that is essential to induce flowering specifically under SD conditions in Arabidopsis. nfl mutants do not flower under SD conditions, but flower similar to the wild type under LD conditions. The no-flowering phenotype in SD is rescued either by exogenous application of gibberellin (GA) or by introducing della quadruple mutants in the nfl background, suggesting that NFL acts upstream of GA to promote flowering. NFL is expressed at the meristematic regions and NFL is localized to the nucleus. Quantitative RT-PCR assays using apical tissues showed that GA biosynthetic genes are downregulated and the GA catabolic and receptor genes are upregulated in the nfl mutant compared with the wild type, consistent with the perturbation of the endogenous GA biosynthetic and catabolic intermediates in the mutant. Taken together, these data suggest that NFL is a key transcription factor necessary for promotion of flowering under non-inductive SD conditions through the GA signaling pathway.

NbGIS regulates glandular trichome initiation through GA signaling in tobacco.

KEY MESSAGE: A novel gene NbGIS positively regulates glandular trichome initiation through GA Signaling in tobacco. NbMYB123-like regulates glandular trichome initiation by acting downstream of NbGIS in tobacco. Glandular trichome is a specialized multicellular structure which has capability to synthesize and secrete secondary metabolites and protects plants from biotic and abiotic stresses. Our previous results revealed that a C2H2 zinc-finger transcription factor GIS and its sub-family genes act upstream of GL3/EGL3-GL1-TTG1 transcriptional activator complex to regulate trichome initiation in Arabidopsis. In this present study, we found that NbGIS could positively regulate glandular trichome development in Nicotiana benthamiana (tobacco). Our result demonstrated that 35S:NbGIS lines exhibited much higher densities of trichome on leaves, main stems, lateral branches and sepals than WT plants, while NbGIS:RNAi lines had the opposite phenotypes. Furthermore, our results also showed that NbGIS was required in response to GA signal to control glandular trichome initiation in Nicotiana benthamiana. In addition, our results also showed that NbGIS significantly influenced GA accumulation and expressions of marker genes of the GA biosynthesis, might result in the changes of growth and maturation in tobacco. Lastly, our results also showed that NbMYB123-like regulated glandular trichome initiation in tobacco by acting downstream of NbGIS. These findings provide new insights to discover the molecular mechanism by which C2H2 transcriptional factors regulates glandular trichome initiation through GA signaling pathway in tobacco.

Isolation of a novel protein phosphatase2C in rice and its response to gibberellin.

Protein phosphatase 2Cs (PP2Cs) have been referred to act as negative modulators of the protein kinase pathways involved in different environmental stress responses and developmental processes. In Arabidopsis, PP2Cs have been extensively studied and some are known to negatively regulate abscisic acid signaling. In rice, PP2Cs are scarcely characterized functionally. Here, we identified a novel PP2C from rice (OsPP2C34), which is highly inducible by gibberellin (GA) and expressed in various tissues. Subcellular localization analysis in maize protoplasts using a green fluorescence protein fusion vector localized OsPP2C34 to the cytosol. Genetic analysis of T-DNA insertional mutants revealed that plant height and internode length were significantly shorter in mutants than in corresponding wild types under GA treatment. The induction of the GA-inducibleα-amylase genes RAmy3E and OsAmy was delayed in mutant plants. The substrate of OsPP2C34 was identified by immunoblotting using anti serine/threonine antibodies. A 65 kDa protein was phosphorylated in Ospp2c34-1 but dephosphorylated in the wild type during early germination stage. Overall, the present results indicated that OsPP2C34 is involved inα-amylase expression of GA signal transduction pathway.

Plant Stature Related receptor-like Kinanse2 (PSRK2) acts as a factor that determines stem elongation toward gibberellins response in rice.

Gibberellins (GAs) are a family of plant hormones that are important to multiple aspects of plant growth and development, especially stem elongation. A PSRK2 was obtained through screening and identifying RLK dominant negative mutants. Phenotype of the loss-of-function mutants, psrk2-DN and psrk2-RNAi, showed that PSRK2 could influence the length of the uppermost and fourth internodes, indicating that PSRK2 might regulate cell division in the intercalary meristems and/or cell elongation in the internodes. Moreover, the expression pattern showed that PSRK2 was strongly expressed in the joined-nodes after the start-up of reproductive growth, but undetectable in leaves. PSRK2 expression was also found to be induced by GA3, and PSRK2 was involved in GA signaling in cereal aleurone cells, and PSRK2 influence the relative length of the second leaf sheaths in seedling stage. These results indicate PSRK2 is a component of GA signaling pathway that controls stem elongation by negatively regulating GA responses. Abbreviations: Os: Oryza sativa; At: Arabidopsis thaliana; RNAi: RNA interfere; DN: Dominate Negative; SMART: Simple Modular Architecture Research Tool; Uni : Uniconazol; PSRK2: Plant Stature Related receptor-like Kinase 2; RLK: Receptor-like Kinase; GA: Gibberellin; IAA: indole-3-acetic acid; BL: Brassinosteroid.

AtGIS, a C2H2 zinc-finger transcription factor from Arabidopsis regulates glandular trichome development through GA signaling in tobacco.

Glandular trichome is specialized multicellular structures that have capability to synthesize and secrete secondary metabolites and protect plants from biotic and abiotic stresses. Our previous results revealed that the C2H2 zinc-finger transcription factors (GIS) acts upstream of GL3/EGL3-GL1-TTG1transcriptional activator complex to regulate trichome initiation through GA signal in Arabidopsis. In the present study, we are reporting that ectopic expression of AtGIS could regulate glandular trichome development through GA signaling in tobacco. X-gluc staining of various organs from transgenic plants showed that AtGIS expressed mainly in the glandular trichomes. Statistical analysis demonstrated that over expression of GIS increased significantly glandular trichome production on the leaf, stem, branch, and sepal in tobacco. After PAC treatment, reduction of glandular trichome production in transgenic plants was more severe with compared to wild type plants. Furthermore, GA treatment could induce expression of AtGIS. More importantly, our results also demonstrated that overexpressed AtGIS significantly affect the main components of trichome exudates, such as significantly increase the content of nicotine, Cembratriene-4, 6-diol. Taken together, these results suggest that ectopic expression of AtGIS regulates glandular trichome development and may play a key role in compounds secretion in tobacco.

A fungal endophyte helps plants to tolerate root herbivory through changes in gibberellin and jasmonate signaling.

Plant-microbe mutualisms can improve plant defense, but the impact of root endophytes on below-ground herbivore interactions remains unknown. We investigated the effects of the root endophyte Piriformospora indica on interactions between rice (Oryza sativa) plants and its root herbivore rice water weevil (RWW; Lissorhoptrus oryzophilus), and how plant jasmonic acid (JA) and GA regulate this tripartite interaction. Glasshouse experiments with wild-type rice and coi1-18 and Eui1-OX mutants combined with nutrient, jasmonate and gene expression analyses were used to test: whether RWW adult herbivory above ground influences subsequent damage caused by larval herbivory below ground; whether P. indica protects plants against RWW; and whether GA and JA signaling mediate these interactions. The endophyte induced plant tolerance to root herbivory. RWW adults and larvae acted synergistically via JA signaling to reduce root growth, while endophyte-elicited GA biosynthesis suppressed the herbivore-induced JA in roots and recovered plant growth. Our study shows for the first time the impact of a root endophyte on plant defense against below-ground herbivores, adds to growing evidence that induced tolerance may be an important root defense, and implicates GA as a signal component of inducible plant tolerance against biotic stress.

Expression and purification of a GRAS domain of SLR1, the rice DELLA protein.

GRAS proteins belong to a plant specific protein family that participates in diverse and important functions in growth and development. GRAS proteins are typically composed of a variable N-terminal domain and highly conserved C-terminal GRAS domain. Despite the importance of the GRAS domain, little biochemical or structural analyses have been reported, mainly due to difficulties with purification of sufficient quality and quantity of protein. This study is focused on one of the most extensively studied GRAS proteins, the rice DELLA protein (SLR1), which is known to be involved in gibberellin (GA) signaling. Using a baculovirus-insect cell expression system we have achieved overproduction and purification of full-length SLR1. Limited proteolysis of the full-length SLR1 indicated that a region including the entire GRAS domain (SLR1(206-625)) is protease resistant. Based on those results, we have constructed an expression and purification system of the GRAS domain (SLR1(206-625)) in Escherichia coli. Several physicochemical assays have indicated that the folded structure of the GRAS domain is rich in secondary structural elements and that alanine substitutions for six cysteine residues improves protein folding without impairing function. Furthermore, by NMR spectroscopy we have observed direct interaction between the purified GRAS domain and the GA receptor GID1. Taken together, our purified preparation of the GRAS domain of SLR1 is suitable for further structural and functional studies that will contribute to precise understanding of the plant regulation mechanism through DELLA and GRAS proteins.

Transcription factor TERF1 promotes seed germination under osmotic conditions by activating gibberellin acid signaling.

Seed germination is the first step of seedling establishment, which is particularly sensitive to drought stress. Elucidating the mechanism regulating seed germination under drought stress is of great importance. We showed that overexpressing Tomato Ethylene Responsive Factor 1 (TERF1), an ERF transcription factor in the ethylene signaling pathway, significantly reduced seed sensitivity to mannitol treatment during seed germination. Germination assay demonstrated that TERF1 could activate gibberellin acid (GA) signaling pathway independent on GA metabolism during germination. By comparative transcriptome analysis (mannitol vs normal germination condition, mannitol vs mannitol plus paclobutrazol (PAC, an inhibitor of GA biosynthesis)) we identified the genes regulated by TERF1 specifically under mannitol treatment and confirmed that TERF1 could activate GA signaling pathway independent on GA metabolism, which were consistent with the germination assay with mannitol and mannitol plus PAC treatment. Based on sugar, gene expression and germination analysis we proved that TERF1 promoted seed germination through glucose signaling pathway mediated by GA. Thus our study provides an underlying mechanism for activating GA signaling pathway by TERF1 during seed germination under osmotic conditions.