CUP-SHAPED COTYLEDON Research Articles

Construction of vector and transformation of drought-responsive gene OsNAC1 into J02 (Oryza sativa L. Japonica) rice

NAC (including NAM - no apical meristem, ATAF1/2 - Arabidopsis transcription activation factor and CUC2 - cup-shaped cotyledon), which is the largest plant transcription factor family, plays an important role in development and stress responses in plants. Protein of this family is characterized by a highly conserved DNA binding domain, known as NAC domain in the N-terminal region. In contrast, the C-terminal region of NAC proteins, usually containing the transcriptional activation domain, is highly diversified both in length and sequence. More than 100 members of this family have been identified in rice. However, only a few of them have been functionally characterized, especially in rice. Gene encoding transcription factor OsNAC1 has been proved to play an important role in drought stress in plants. The CaMV35S promoter derived from the common plant virus, cauliflower mosaic virus (CaMV), is a component of transgenic constructs in more than 80% of genetically modified (GM) plants. It is the promoter of choice for plant genetic engineering, as it is a strong and constitutive promoter. In this study, an expression vector harboring OsNAC1 in the form of CaMV35S:OsNAC1:Nos was constructed and transferred into J02 (Oryza sativa L. Japonica) rice plants via Agrobacterium tumefaciens. The presence of the transgene was confirmed by PCR using OsNAC1 specific primers. T0 CaMV35S:OsNAC1:Nos transgenic lines were selected from transgenic plants. The obatained results are expected to be further exploited for development of stress tolerant rice varieties in the future.

Over-expression of a NAC 67 transcription factor from finger millet (Eleusine coracana L.) confers tolerance against salinity and drought stress in rice.

BackgroundNAC proteins (NAM (No apical meristem), ATAF (Arabidopsis transcription activation factor) and CUC (cup-shaped cotyledon)) are plant-specific transcription factors reported to be involved in regulating growth, development and stress responses. Salinity responsive transcriptome profiling in a set of contrasting finger millet genotypes through RNA-sequencing resulted in the identification of a NAC homolog (EcNAC 67) exhibiting differential salinity responsive expression pattern.MethodsFull length cDNA of EcNAC67 was isolated, characterized and validated for its role in abiotic stress tolerance through agrobacterium mediated genetic transformation in a rice cultivar ASD16.ResultsBioinformatics analysis of putative NAC transcription factor (TF) isolated from a salinity tolerant finger millet showed its genetic relatedness to NAC67 family TFs in related cereals. Putative transgenic lines of rice over-expressing EcNAC67 were generated through Agrobacterium mediated transformation and presence/integration of transgene was confirmed through PCR and southern hybridization analysis. Transgenic rice plants harboring EcNAC67 showed enhanced tolerance against drought and salinity under greenhouse conditions. Transgenic rice plants were found to possess higher root and shoot biomass during stress and showed better revival ability upon relief from salinity stress. Upon drought stress, transgenic lines were found to maintain higher relative water content and lesser reduction in grain yield when compared to non-transgenic ASD16 plants. Drought induced spikelet sterility was found to be much lower in the transgenic lines than the non-transgenic ASD16.ConclusionResults revealed the significant role of EcNAC67 in modulating responses against dehydration stress in rice. No detectable abnormalities in the phenotypic traits were observed in the transgenic plants under normal growth conditions. Results indicate that EcNAC67 can be used as a novel source for engineering tolerance against drought and salinity stress in rice and other crop plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0261-1) contains supplementary material, which is available to authorized users.

REF6 recognizes a specific DNA sequence to demethylate H3K27me3 and regulate organ boundary formation in Arabidopsis.

RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) counteracts Polycomb-mediated gene silencing by removing methyl groups from trimethylated histone H3 lysine 27 (H3K27me3) in hundreds of genes in Arabidopsis thaliana. Here we show that REF6 function and genome-wide targeting require its four Cys2His2 zinc fingers, which directly recognize a CTCTGYTY motif. Motifs bound by REF6 tend to cluster and reside in loci with active chromatin states. Furthermore, REF6 targets CUP-SHAPED COTYLEDON 1 (CUC1), which harbors CTCTGYTY motifs, to modulate H3K27me3 levels and activate CUC1 expression. Loss of REF6 causes CUC1 repression and defects in cotyledon separation. In contrast, REF6 does not bind CUC2, encoding a close homolog of CUC1, which lacks the CTCTGYTY motif. Collectively, these results identify a new targeting mechanism of an H3K27 demethylase to counteract Polycomb-mediated gene silencing that regulates plant development, including organ boundary formation.

A Conserved Role for the NAM/miR164 Developmental Module Reveals a Common Mechanism Underlying Carpel Margin Fusion in Monocarpous and Syncarpous Eurosids.

The majority of angiosperms are syncarpous- their gynoecium is composed of two or more fused carpels. In Arabidopsis thaliana, this fusion is regulated through the balance of expression between CUP SHAPED COTYLEDON (CUC) genes, which are orthologs of the Petunia hybrida transcription factor NO APICAL MERISTEM (NAM), and their post-transcriptional regulator miR164. Accordingly, the expression of a miR164-insensitive form of A. thaliana CUC2 causes a radical breakdown of carpel fusion. Here, we investigate the role of the NAM/miR164 genetic module in carpel closure in monocarpous plants. We show that the disruption of this module in monocarpous flowers of A. thaliana aux1-22 mutants causes a failure of carpel closure, similar to the failure of carpel fusion observed in the wild-type genetic background. This observation suggested that closely related mechanisms may bring about carpel closure and carpel fusion, at least in A. thaliana. We therefore tested whether these mechanisms were conserved in a eurosid species that is monocarpous in its wild-type form. We observed that expression of MtNAM, the NAM ortholog in the monocarpous eurosid Medicago truncatula, decreases during carpel margin fusion, suggesting a role for the NAM/miR164 module in this process. We transformed M. truncatula with a miR164-resistant form of MtNAM and observed, among other phenotypes, incomplete carpel closure in the resulting transformants. These data confirm the underlying mechanistic similarity between carpel closure and carpel fusion which we observed in A. thaliana. Our observations suggest that the role of the NAM/miR164 module in the fusion of carpel margins has been conserved at least since the most recent common ancestor of the eurosid clade, and open the possibility that a similar mechanism may have been responsible for carpel closure at much earlier stages of angiosperm evolution. We combine our results with studies of early diverging angiosperms to speculate on the role of the NAM/miR164 module in the origin and further evolution of the angiosperm carpel.

Cloning and characterization of drought stress-induced NAC transcription factors fromBrassica junceaandSinapis alba

The plant specific NAC [for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)] TFs are one of the largest plant TF families that play important roles in plant development and stress tolerance. Suppression subtractive hybridization (SSH) analysis with using drought stressed plants of S. alba lead to the identification of several stress responsive ESTs. Two of them homologous to Arabidopsis NAC14 and NAC19 were selected for cloning of full length CDS and expression analysis in Brassica and related species with contrasting drought tolerance. NAC14 and NAC19 genes were cloned from drought tolerant Sinapis alba and Brassica juncea cvs. RGN73 and Varuna, and drought sensitive B. juncea cvs. RLM619, BEC144 and BioYSR. Sequencing of genomic region coding for these NACs revealed that both NAC14 and NAC19 contain 3 exons and 2 introns each. In silico analysis of protein structure led to development of 3D models of these stress responsive NAC TFs. Although both proteins have 7 strands of β sheets, the NAC14 had 5 β sheets of type A and 2 β sheets of type B, while NAC14 have all 7 β sheets of type A. These proteins also differed in helix content, β turns and g turns. This suggest their functional diversity under abiotic stresses. Real-time RT-PCR expression analysis revealed that both the genes were up-regulated under drought stress in the leaves of B. juncea genotypes Varuna and BioYSR. In addition, NAC14 was up-regulated in the leaves of RLM619, while NAC19 was up-regulated in the leaves of S. alba and BEC144 under drought stress as compared to control conditions. Interestingly, drought stress did not up-regulate these genes in RGN73. This study revealed genotypic variation in the drought regulation of NAC TFs in B. juncea and S. alba.

Genome-Wide Identification and Expression Analysis of the NAC Transcription Factor Family in Cassava.

NAC [no apical meristem (NAM), Arabidopsis transcription activation factor [ATAF1/2] and cup-shaped cotyledon (CUC2)] proteins is one of the largest groups of plant specific transcription factors and plays a crucial role in plant growth, development, and adaption to the environment. Currently, no information is known about the NAC family in cassava. In this study, 96 NAC genes (MeNACs) were identified from the cassava genome. Phylogenetic analysis of the NACs from cassava and Arabidopsis showed that MeNAC proteins can be clustered into 16 subgroups. Gene structure analysis found that the number of introns of MeNAC genes varied from 0 to 5, with the majority of MeNAC genes containing two introns, indicating a small gene structure diversity of cassava NAC genes. Conserved motif analysis revealed that all of the identified MeNACs had the conserved NAC domain and/or NAM domain. Global expression analysis suggested that MeNAC genes exhibited different expression profiles in different tissues between wild subspecies and cultivated varieties, indicating their involvement in the functional diversity of different accessions. Transcriptome analysis demonstrated that MeNACs had a widely transcriptional response to drought stress and that they had differential expression profiles in different accessions, implying their contribution to drought stress resistance in cassava. Finally, the expression of twelve MeNAC genes was analyzed under osmotic, salt, cold, ABA, and H2O2 treatments, indicating that cassava NACs may represent convergence points of different signaling pathways. Taken together, this work found some excellent tissue-specific and abiotic stress-responsive candidate MeNAC genes, which would provide a solid foundation for functional investigation of the NAC family, crop improvement and improved understanding of signal transduction in plants. These data bring new insight on the complexity of the transcriptional control of MeNAC genes and support the hypothesis that NACs play an important role in plant growth, development, and adaption of environment.

A conserved role for CUP-SHAPED COTYLEDON genes during ovule development.

The evolution of plant reproductive strategies has led to a remarkable diversity of structures, especially within the flower, a structure characteristic of the angiosperms. In flowering plants, sexual reproduction depends notably on the development of the gynoecium that produces and protects the ovules. In Arabidopsis thaliana, ovule initiation is promoted by the concerted action of auxin with CUC1 (CUP-SHAPED COTYLEDON1) and CUC2, two genes that encode transcription factors of the NAC family (NAM/ATAF1,2/CUC). Here we highlight an additional role for CUC2 and CUC3 in Arabidopsis thaliana ovule separation. While CUC1 and CUC2 are broadly expressed in the medial tissue of the gynoecium, CUC2 and CUC3 are expressed in the placental tissue between developing ovules. Consistent with the partial overlap between CUC1, CUC2 and CUC3 expression patterns, we show that CUC proteins can physically interact, both in yeast cells and in planta. We found that the cuc2;cuc3 double mutant specifically harbours defects in ovule separation, producing fused seeds that share the seed coat, and suggesting that CUC2 and CUC3 promote ovule separation in a partially redundant manner. Functional analyses show that CUC transcription factors are also involved in ovule development in Cardamine hirsuta. Additionally we show a conserved expression pattern of CUC orthologues between ovule primordia in other phylogenetically distant species with different gynoecium architectures. Taken together these results suggest an ancient role for CUC transcription factors in ovule separation, and shed light on the conservation of mechanisms involved in the development of innovative structures.

A putative NAP homolog specifically expressed during grapevine flower and berry development

Introduction: In flowering plants, fruit morphogenesis is a distinct and unique process following fertilization. Most fleshy fruits as grapevine develop from ovary tissues (EAMES and MACDANIELS 1947) and exhibit convergent characteristics such as pericarp cell proliferation and enlargement. The early molecular events that control the dynamics of fruit morphogenesis remain obscure. From preliminary analysis of the fleshless berry mutant (Vitis vinifera L. cv. Ugni Blanc) which is unable to specify ovary cell fate later in fleshy pericarp development, we have identified a gene homolog of PISTILLATA up-regulated in the mutant ovary. In Arabidopsis, NAP (AJ222713) appeared as an immediate target of the floral homeotic genes PISTILLATA/APETALA3 (SLABLOWSKI and MEYEROWITZ 1998). NAP genes belong to the NAC domain gene family encoding transcriptional regulators required for meristem establishment and other plant developmental processes. The original genes NO APICAL MERISTEM (NAM) from petunia, ATAF1, 2 and CUPSHAPED COTYLEDON (CUC2) from Arabidopsis defined the highly conserved NAC protein domain (AIDA et al. 1997). We report, here, quantitative expression patterns of the closest NAP grapevine homolog (TC38700 from the TIGR database), VvNAP, during grapevine development of the flb mutant and the wild type genotypes. Material and Methods: The flb mutation was identified in 1996 in a vineyard located at Prades-le-Lez (France) on a Vitis vinifera cv. Ugni-Blanc anticlinal chimeric plant bearing some shoots with berries dramatically reduced in size. For flb mutant and wild-type Ugni Blanc, samples corresponded to inflorescences from stage 17 (mature inflorescence with all flowers formed and separated) and 19 (first flower caps loosening) of the E-L system (COOMBE 1995) and berries from 0 to 20 d after anthesis collected in the greenhouse (ENSA.M-INRA, Montpellier). For V. vinifera cv. Cabernet-Sauvignon, samples corresponded to vegetative organs (young roots, shoot tips including 2 to 3 expanded leaves and young leaves), inflorescences from stages 7 (immature inflorescence emerging from buds) and 17, flowers at flowering time (50 % caps off) and seedless berries (2-16 weeks post flowering), harvested in the field (Clare Valley, Australia). Total RNA was extracted using RNeasy Plant Mini kit (Qiagen) for Ugni Blanc and according to REZAIAN and KRAKE (1987) with slight modifications as described in BOSS et al. (2001) for Cabernet-Sauvignon.

PLETHORA Genes Control Regeneration by a Two-Step Mechanism

Regeneration, a remarkable example of developmental plasticity displayed by both plants and animals, involves successive developmental events driven in response to environmental cues. Despite decades of study on the ability of the plant tissues to regenerate a complete fertile shoot system after inductive cues, the mechanisms by which cells acquire pluripotency and subsequently regenerate complete organs remain unknown. Here, we show that three PLETHORA (PLT) genes, PLT3, PLT5, and PLT7, regulate de novo shoot regeneration in Arabidopsis by controlling two distinct developmental events. Cumulative loss of function of these three genes causes the intermediate cell mass, callus, to be incompetent to form shoot progenitors, whereas induction of PLT5 or PLT7 can render shoot regeneration hormone-independent. We further show that PLT3, PLT5, and PLT7 establish pluripotency by activating root stem cell regulators PLT1 and PLT2, as reconstitution of either PLT1 or PLT2 in the plt3; plt5-2; plt7 mutant re-established the competence to regenerate shoot progenitor cells but did not lead to the completion of shoot regeneration. PLT3, PLT5, and PLT7 additionally regulate and require the shoot-promoting factor CUP-SHAPED COTYLEDON2 (CUC2) to complete the shoot-formation program. Our findings uncouple the acquisition of competence to regenerate shoot progenitor cells from completion of shoot formation, indicating a two-step mechanism of de novo shoot regeneration that operates in all tested plant tissues irrespective of their origin. Our studies reveal intermediate developmental phases of regeneration and provide a deeper understanding into the mechanistic basis of regeneration.

Modular organization of the white spruce (Picea glauca) transcriptome reveals functional organization and evolutionary signatures.

Summary Transcript profiling has shown the molecular bases of several biological processes in plants but few studies have developed an understanding of overall transcriptome variation. We investigated transcriptome structure in white spruce (Picea glauca), aiming to delineate its modular organization and associated functional and evolutionary attributes.Microarray analyses were used to: identify and functionally characterize groups of co‐expressed genes; investigate expressional and functional diversity of vascular tissue preferential genes which were conserved among Picea species, and identify expression networks underlying wood formation.We classified 22 857 genes as variable (79%; 22 coexpression groups) or invariant (21%) by profiling across several vegetative tissues. Modular organization and complex transcriptome restructuring among vascular tissue preferential genes was revealed by their assignment to coexpression groups with partially overlapping profiles and partially distinct functions. Integrated analyses of tissue‐based and temporally variable profiles identified secondary xylem gene networks, showed their remodelling over a growing season and identified PgNAC‐7 (no apical meristerm (NAM), Arabidopsis transcription activation factor (ATAF) and cup‐shaped cotyledon (CUC) transcription factor 007 in Picea glauca) as a major hub gene specific to earlywood formation.Reference profiling identified comprehensive, statistically robust coexpressed groups, revealing that modular organization underpins the evolutionary conservation of the transcriptome structure.

Natural variation in monoterpene synthesis in kiwifruit: transcriptional regulation of terpene synthases by NAC and ETHYLENE-INSENSITIVE3-like transcription factors.

Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-D-erythritol 4-phosphate pathway enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-D-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits.

Genetic interaction between GROWTH-REGULATING FACTOR and CUP-SHAPED COTYLEDON in organ separation

The Arabidopsis thaliana GROWTH-REGULATING FACTOR (GRF) gene family comprises 9 members and encodes a class of transcription factors. We previously demonstrated that GRF genes played an essential role in formation of the boundary region between cotyledons, since their loss-of-function mutants developed fused cotyledons. Our present study shows that the grf mutants display fused floral organs as well. Such fusion phenotypes of embryonic and post-embryonic floral organs are highly reminiscent of the cup-shaped cotyledon (cuc) mutants. In order to test a genetic interaction between GRFs and CUCs, we constructed cuc1 grf1/2/3, cuc2 grf1/2/3, and cuc3 grf1/2/3 quadruple mutants, and found that the mutants showed dramatic increases in cotyledon fusion as well as floral organ fusion. The results suggest that the signaling pathway of GRFs may be genetically associated with that of CUCs in the organ separation process.

Protein intrinsic disorder in Arabidopsis NAC transcription factors: transcriptional activation by ANAC013 and ANAC046 and their interactions with RCD1.

Protein ID (intrinsic disorder) plays a significant, yet relatively unexplored role in transcription factors (TFs). In the present paper, analysis of the transcription regulatory domains (TRDs) of six phylogenetically representative, plant-specific NAC [no apical meristem, ATAF (Arabidopsis transcription activation factor), cup-shaped cotyledon] TFs shows that the domains are present in similar average pre-molten or molten globule-like states, but have different patterns of order/disorder and MoRFs (molecular recognition features). ANAC046 (Arabidopsis NAC 046) was selected for further studies because of its simple MoRF pattern and its ability to interact with RCD1 (radical-induced cell death 1). Experiments in yeast and thermodynamic characterization suggest that its single MoRF region is sufficient for both transcriptional activation and interaction with RCD1. The remainder of the large regulatory domain is unlikely to contribute to the interaction, since the domain and truncations thereof have similar affinities for RCD1, which are also similar for ANAC013-RCD1 interactions. However, different enthalpic and entropic contributions to binding were revealed for ANAC046 and ANAC013, suggestive of differences in binding mechanisms. Although substitution of both hydrophobic and acidic residues of the ANAC046 MoRF region abolished binding, substitution of other residues, even with α-helix-breaking proline, was less disruptive. Together, the biophysical analyses suggest that RCD1-ANAC046 complex formation does not involve folding-upon-binding, but rather fuzziness or an unknown structure in ANAC046. We suggest that the ANAC046 regulatory domain functions as an entropic chain with a terminal hot spot interacting with RCD1. RCD1, a cellular hub, may be able to interact with many different TFs by exploiting their ID-based flexibility, as demonstrated for its interactions with ANAC046 and ANAC013.

Origination of asexual plantlets in three species of Crassulaceae.

During asexual plant reproduction, cells from different organs can be reprogrammed to produce new individuals, a process that requires the coordination of cell cycle reactivation with the acquisition of other cellular morphological characteristics. However, the factors that influence the variety of asexual reproduction have not yet been determined. Here, we report on plantlet formation in Kalanchoe daigremontiana, Graptopetalum paraguayense, and Crassula portulacea (Crassulaceae) and analyse the effect of initiating cells on asexual reproduction in these three species. Additionally, the roles of WUSCHEL (WUS) and CUP-SHAPED COTYLEDON 1 (CUC1) in the asexual reproduction of these species were analysed through qRT-PCR. Our results indicated that pre-existing stem cell-like cells at the sites of asexual reproduction were responsible for the formation of plantlets. These cells were arrested in different phases of the cell cycle and showed different cell morphological characteristics and cell counts. The accumulation of auxin and cytokinin at the sites of asexual plantlet formation indicated their important functions, particularly for cell cycle reactivation. These differences may influence the pattern and complexity of asexual reproduction in these Crassulaceae species. Additionally, the dynamic expression levels of CUC1 and WUS may indicate that CUC1 functions in the formation of callus and shoot meristems; whereas, WUS was only associated with shoot induction.

Transcriptome profiling reveals roles of meristem regulators and polarity genes during fruit trichome development in cucumber (Cucumis sativus L.).

Trichomes are epidermal hair-like structures that function in plant defence against biotic and abiotic stresses. Extensive studies have been performed on foliar trichomes development in Arabidopsis and tomato, but the molecular mechanism of fruit trichome formation remains elusive. Cucumber fruit is covered with trichomes (spines) that directly affect the appearance and quality of cucumber products. Here, we characterized the fruit spine development in wild-type (WT) cucumber and a spontaneous mutant, tiny branched hair (tbh). Our data showed that the cucumber trichome was multicellular and non-glandular, with malformed organelles and no endoreduplication. Fruit spine development was generally homogenous and marked by a rapid base expansion stage. Trichomes in the tbh mutant were tiny and branched, with increased density and aberrant cell shape. Transcriptome profiling indicated that meristem-related genes were highly enriched in the upregulated genes in the tbh versus the WT, as well as in WT spines after versus before base expansion, and that polarity regulators were greatly induced during spine base expansion. Quantitative reverse transcription PCR and in situ hybridization confirmed the differential expression of CUP-SHAPED COTYLEDON3 (CUC3) and SHOOT MERISTEMLESS (STM) during spine development. Therefore, cucumber trichomes are morphologically different from those of Arabidopsis and tomato, and their development may be regulated by a distinct pathway involving meristem genes and polarity regulators.

OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice.

It has long been established that premature leaf senescence negatively impacts the yield stability of rice, but the underlying molecular mechanism driving this relationship remains largely unknown. Here, we identified a dominant premature leaf senescence mutant, prematurely senile 1 (ps1-D). PS1 encodes a plant-specific NAC (no apical meristem, Arabidopsis ATAF1/2, and cup-shaped cotyledon2) transcriptional activator, Oryza sativa NAC-like, activated by apetala3/pistillata (OsNAP). Overexpression of OsNAP significantly promoted senescence, whereas knockdown of OsNAP produced a marked delay of senescence, confirming the role of this gene in the development of rice senescence. OsNAP expression was tightly linked with the onset of leaf senescence in an age-dependent manner. Similarly, ChIP-PCR and yeast one-hybrid assays demonstrated that OsNAP positively regulates leaf senescence by directly targeting genes related to chlorophyll degradation and nutrient transport and other genes associated with senescence, suggesting that OsNAP is an ideal marker of senescence onset in rice. Further analysis determined that OsNAP is induced specifically by abscisic acid (ABA), whereas its expression is repressed in both aba1 and aba2, two ABA biosynthetic mutants. Moreover, ABA content is reduced significantly in ps1-D mutants, indicating a feedback repression of OsNAP on ABA biosynthesis. Our data suggest that OsNAP serves as an important link between ABA and leaf senescence. Additionally, reduced OsNAP expression leads to delayed leaf senescence and an extended grain-filling period, resulting in a 6.3% and 10.3% increase in the grain yield of two independent representative RNAi lines, respectively. Thus, fine-tuning OsNAP expression should be a useful strategy for improving rice yield in the future.

LEAFY and Polar Auxin Transport Coordinately Regulate Arabidopsis Flower Development.

The plant specific transcription factor LEAFY (LFY) plays a pivotal role in the developmental switch to floral meristem identity in Arabidopsis. Our recent study revealed that LFY additionally acts downstream of AUXIN RESPONSE FACTOR5/MONOPTEROS to promote flower primordium initiation. LFY also promotes initiation of the floral organ and floral organ identity. To further investigate the interplay between LFY and auxin during flower development, we examined the phenotypic consequence of disrupting polar auxin transport in lfy mutants by genetic means. Plants with compromised LFY activity exhibit increased sensitivity to disruption of polar auxin transport. Compromised polar auxin transport activity in the lfy mutant background resulted in formation of fewer floral organs, abnormal gynoecium development, and fused sepals. In agreement with these observations, expression of the auxin response reporter DR5rev::GFP as well as of the direct LFY target CUP-SHAPED COTYLEDON2 were altered in lfy mutant flowers. We also uncovered reduced expression of ETTIN, a regulator of gynoecium development and a direct LFY target. Our results suggest that LFY and polar auxin transport coordinately modulate flower development by regulating genes required for elaboration of the floral organs.

The CUC1 and CUC2 genes promote carpel margin meristem formation during Arabidopsis gynoecium development.

Carpel margin meristems (CMMs), a pair of meristematic tissues present along the margins of two fused carpel primordia of Arabidopsis thaliana, are essential for the formation of ovules and the septum, two major internal structures of the gynoecium. Although a number of regulatory factors involved in shoot meristem activity are known to be required for the formation of these gynoecial structures, their direct roles in CMM development have yet to be addressed. Here we show that the CUP-SHAPED COTYLEDON genes CUC1 and CUC2, which are essential for shoot meristem initiation, are also required for formation and stable positioning of the CMMs. Early in CMM formation, CUC1 and CUC2 are also required for expression of the SHOOT MERISTEMLESS gene, a central regulator for stem cell maintenance in the shoot meristem. Moreover, plants carrying miR164-resistant forms of CUC1 and CUC2 resulted in extra CMM activity with altered positioning. Our results thus demonstrate that the two regulatory proteins controlling shoot meristem activity also play critical roles in elaboration of the female reproductive organ through the control of meristematic activity.

Differential activation of sporamin expression in response to abiotic mechanical wounding and biotic herbivore attack in the sweet potato.

BackgroundPlants respond differently to mechanical wounding and herbivore attack, using distinct pathways for defense. The versatile sweet potato sporamin possesses multiple biological functions in response to stress. However, the regulation of sporamin gene expression that is activated upon mechanical damage or herbivore attack has not been well studied.ResultsBiochemical analysis revealed that different patterns of Reactive oxygen species (ROS) and antioxidant mechanism exist between mechanical wounding (MW) and herbivore attack (HA) in the sweet potato leaf. Using LC-ESI-MS (Liquid chromatography electrospray ionization mass spectrometry analysis), only the endogenous JA (jasmonic acid) level was found to increase dramatically after MW in a time-dependent manner, whereas both endogenous JA and SA (salicylic acid) increase in parallel after HA. Through yeast one-hybrid screening, two transcription factors IbNAC1 (no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC)) and IbWRKY1 were isolated, which interact with the sporamin promoter fragment of SWRE (sporamin wounding-responsive element) regulatory sequences. Exogenous application of MeJA (methyl jasmonate), SA and DIECA (diethyldithiocarbamic acid, JAs biosynthesis inhibitor) on sweet potato leaves was employed, and the results revealed that IbNAC1 mediated the expression of sporamin through a JA-dependent signaling pathway upon MW, whereas both IbNAC1 and IbWRKY1 coordinately regulated sporamin expression through JA- and SA-dependent pathways upon HA. Transcriptome analysis identified MYC2/4 and JAZ2/TIFY10A (jasmonate ZIM/tify-domain), the repressor and activator of JA and SA signaling among others, as the genes that play an intermediate role in the JA and SA pathways, and these results were further validated by qRT-PCR (quantitative real-time polymerase chain reaction).ConclusionThis work has improved our understanding of the differential regulatory mechanism of sporamin expression. Our study illustrates that sweet potato sporamin expression is differentially induced upon abiotic MW and biotic HA that involves IbNAC1 and IbWRKY1 and is dependent on the JA and SA signaling pathways. Thus, we established a model to address the plant-wounding response upon physical and biotic damage.

Involvement of rRNA biosynthesis in the regulation of CUC1 gene expression and pre-meristematic cell mound formation during shoot regeneration.

At an early stage of shoot regeneration from calli of Arabidopsis, pre-meristematic cell mounds develop in association with localized strong expression of CUP-SHAPED COTYLEDON (CUC) genes. Previous characterization of root initiation-defective 3 (rid3), an Arabidopsis mutant originally isolated as being temperature-sensitive for adventitious root formation, with respect to shoot regeneration implicated RID3 in the negative regulation of CUC1 expression and the restriction of cell division in pre-meristematic cell mounds. Positional cloning has identified RID3 as a WD40 repeat protein gene whose molecular function was not investigated before. Here we performed in silico analysis of RID3 and found that RID3 is orthologous to IPI3, which mediates pre-rRNA processing in Saccharomyces cerevisiae. In the rid3 mutant, rRNA precursors accumulated to a very high level in a temperature-dependent manner. This result indicates that RID3 is required for pre-rRNA processing as is IPI3. We compared rid3 with rid2, a temperature-sensitive mutant that is mutated in a putative RNA methyltransferase gene and is impaired in pre-rRNA processing, for seedling morphology, shoot regeneration, and CUC1 expression. The rid2 and rid3 seedlings shared various developmental alterations, such as a pointed-leaf phenotype, which is often observed in ribosome-related mutants. In tissue culture for the induction of shoot regeneration, both rid2 and rid3 mutations perturbed cell-mound formation and elevated CUC1 expression. Together, our findings suggest that rRNA biosynthesis may be involved in the regulation of CUC1 gene expression and pre-meristematic cell-mound formation during shoot regeneration.