Application of ethanol alleviates heat damage to leaf growth and yield in tomato.

Chemical priming has emerged as a promising area in agricultural research. Our previous studies have demonstrated that pretreatment with a low concentration of ethanol enhances abiotic stress tolerance in Arabidopsis and cassava. Here, we show that ethanol treatment induces heat stress tolerance in tomato (Solanum lycopersicon L.) plants. Seedlings of the tomato cultivar 'Micro-Tom' were pretreated with ethanol solution and then subjected to heat stress. The survival rates of the ethanol-pretreated plants were significantly higher than those of the water-treated control plants. Similarly, the fruit numbers of the ethanol-pretreated plants were greater than those of the water-treated ones. Transcriptome analysis identified sets of genes that were differentially expressed in shoots and roots of seedlings and in mature green fruits of ethanol-pretreated plants compared with those in water-treated plants. Gene ontology analysis using these genes showed that stress-related gene ontology terms were found in the set of ethanol-induced genes. Metabolome analysis revealed that the contents of a wide range of metabolites differed between water- and ethanol-treated samples. They included sugars such as trehalose, sucrose, glucose, and fructose. From our results, we speculate that ethanol-induced heat stress tolerance in tomato is mainly the result of increased expression of stress-related genes encoding late embryogenesis abundant (LEA) proteins, reactive oxygen species (ROS) elimination enzymes, and activated gluconeogenesis. Our results will be useful for establishing ethanol-based chemical priming technology to reduce heat stress damage in crops, especially in Solanaceae.

NAC domain transcription factors VNI2 and ATAF2 form protein complexes and regulate leaf senescence.

The NAM, ATAF1/2, and CUC2 (NAC) domain transcription factor VND-INTERACTING2 (VNI2) negatively regulates xylem vessel formation by interacting with another NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), a master regulator of xylem vessel formation. Here, we screened interacting proteins with VNI2 using yeast two-hybrid assay and isolated two NAC domain transcription factors, Arabidopsis thaliana ACTIVATION FACTOR 2 (ATAF2) and NAC DOMAIN CONTAINING PROTEIN 102 (ANAC102). A transient gene expression assay showed that ATAF2 upregulates the expression of genes involved in leaf senescence, and VNI2 effectively inhibits the transcriptional activation activity of ATAF2. vni2 mutants accelerate leaf senescence, whereas ataf2 mutants delay leaf senescence. In addition, the accelerated leaf senescence phenotype of the vni2 mutant is recovered by simultaneous mutation of ATAF2. Our findings strongly suggest that VNI2 interacts with and inhibits ATAF2, resulting in negatively regulating leaf senescence.

Genome editing and molecular analyses of an Arabidopsis transcription factor, LATE FLOWERING.

Correct flower organ formation at the right timing is one of the most important strategies for plants to achieve reproductive success. Ectopic overexpression of LATE FLOWERING (LATE) is known to induce late flowering, partly through suppressing expression of the florigen-encoding gene FLOWERING LOCUS T (FT) in Arabidopsis. LATE is one of the C2H2 zinc finger transcription factors, and it has a canonical transcriptional repression domain called the ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif at the end of its C terminus. Therefore, LATE is considered a transcriptional repressor, but its molecular function remains unclear. Our genome-edited late mutants exhibited no distinct phenotype, even in flowering, indicating the presence of redundancy from other factors. To reveal the molecular function of LATE and factors working with it, we investigated its transcriptional activity and interactions with other proteins. Transactivation activity assay showed that LATE possesses transcriptional repression ability, which appears to be attributable to both the EAR motif and other sequences. Yeast two-hybrid assay showed the EAR motif-mediated interaction of LATE with TOPLESS, a transcriptional corepressor. Moreover, LATE could also interact with CRABS CLAW (CRC), one of the most important regulators of floral meristem determinacy, through sequences in LATE other than the EAR motif. Our findings demonstrated the possibility that LATE can form a transcriptional repression complex with CRC for floral meristem determinacy.

Improving the Efficiency of Adventitious Shoot Induction and Somatic Embryogenesis via Modification of WUSCHEL and LEAFY COTYLEDON 1.

The induction of adventitious organs, such as calli, shoots, and somatic embryos, in tissue culture is a useful technique for plant propagation and genetic modification. In recent years, several genes have been reported to be adventitious organ inducers and proposed to be useful for industrial applications. Even though the Arabidopsis (Arabidopsis thaliana) WUSCHEL (WUS) and LEAFY COTYLEDON 1 (LEC1) genes can induce adventitious organ formation in Arabidopsis without phytohormone treatment, further improvement is desired. Here, we show that modifying the transcriptional repression/activation activities of WUS and LEC1 improves the efficiency of adventitious organ formation in Arabidopsis. Because WUS functions as a transcriptional repressor during the induction of adventitious organs, we fused it to an artificial strong repression domain, SUPERMAN REPRESSION DOMAIN X (SRDX). Conversely, we fused the strong transcriptional activation domain VP16 from herpes simplex virus to LEC1. Upon overexpression of the corresponding transgenes, we succeeded in improving the efficiency of adventitious organ induction. Our results show that the modification of transcriptional repression/activation activity offers an effective method to improve the efficiency of adventitious organ formation in plants.

Holliday Junction Resolvase MOC1 Maintains Plastid and Mitochondrial Genome Integrity in Algae and Bryophytes.

When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination. Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate homologous recombination and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast1 (MOC1) as a plastid DNA HJ resolvase in algae and plants. Although Cruciform cutting endonuclease1 (CCE1) was identified as a mitochondrial DNA HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrate that MOC1 depletion in the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens induced ectopic recombination between short dispersed repeats in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of plastid DNA, MOC1 depletion in P. patens induced short dispersed repeat-mediated ectopic recombination in mitochondrial DNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (RECA2) and mitochondrion-targeted (RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants as well as in plastids in algae and plants.

Author Correction: The Apostasia genome and the evolution of orchids.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Apostasia genome and the evolution of orchids.

Constituting approximately 10% of flowering plant species, orchids (Orchidaceae) display unique flower morphologies, possess an extraordinary diversity in lifestyle, and have successfully colonized almost every habitat on Earth. Here we report the draft genome sequence of Apostasia shenzhenica, a representative of one of two genera that form a sister lineage to the rest of the Orchidaceae, providing a reference for inferring the genome content and structure of the most recent common ancestor of all extant orchids and improving our understanding of their origins and evolution. In addition, we present transcriptome data for representatives of Vanilloideae, Cypripedioideae and Orchidoideae, and novel third-generation genome data for two species of Epidendroideae, covering all five orchid subfamilies. A. shenzhenica shows clear evidence of a whole-genome duplication, which is shared by all orchids and occurred shortly before their divergence. Comparisons between A. shenzhenica and other orchids and angiosperms also permitted the reconstruction of an ancestral orchid gene toolkit. We identify new gene families, gene family expansions and contractions, and changes within MADS-box gene classes, which control a diverse suite of developmental processes, during orchid evolution. This study sheds new light on the genetic mechanisms underpinning key orchid innovations, including the development of the labellum and gynostemium, pollinia, and seeds without endosperm, as well as the evolution of epiphytism; reveals relationships between the Orchidaceae subfamilies; and helps clarify the evolutionary history of orchids within the angiosperms.

WUSCHEL-RELATED HOMEOBOX 2 is a transcriptional repressor involved in lateral organ formation and separation in Arabidopsis.

In this study, we characterized the function of WUSCHEL-RELATED HOMEOBOX 2 (WOX2) using overexpression, CRES-T, and VP16 fusion techniques. Although the function of WOX2 has been described mainly in embryogenesis, it was unclear whether it also plays a role in the post-embryogenic developmental stage. We found that WOX2 has transcriptional repression activity and that either overexpression of WOX2 or expression of its chimeric repressor causes severe growth defects and other morphological phenotypes by impairing plant organ formation and separation. By contrast, VP16-fused WOX2-expressing plants did not display such severe phenotypic defects. In addition, some of them displayed phenotypic defects such as fusion of organs and induction of undifferentiated cells in the boundary regions of organs where GUS staining was clearly observed in the proWOX2:GUS transgenic plants. We suggest that WOX2 is involved in regulation of lateral organ formation and separation during the post-embryogenic development processes.

The ERF transcription factor EPI1 is a negative regulator of dark-induced and jasmonate-stimulated senescence in Arabidopsis.

Identification of the factors involved in the regulation of senescence and the analysis of their function are important for both a biological understanding of the senescence mechanism and the improvement of agricultural productivity. In this study, we identified an ERF gene termed "ERF gene conferring Postharvest longevity Improvement 1" (EPI1) as a possible regulator of senescence in Arabidopsis. We found that EPI1 possesses transcriptional repression activity and that the transgenic plants overexpressing EPI1 and expressing its chimeric repressor, EPI1-SRDX, commonly suppressed the darkness-induced senescence in their excised aerial parts. These transgenic plants additionally maintained a high level of chlorophyll, even after the methyl jasmonate (MeJA) treatment, which stimulated senescence in the dark. In addition, we found that senescence-induced and -reduced genes are down- and upregulated, respectively, in the MeJA-treated transgenic plants under darkness. Our results suggest that EPI1 functions as a negative regulator of the dark-induced and JA-stimulated senescence.

ANAC075, a putative regulator of VASCULAR-RELATED NAC-DOMAIN7, is a repressor of flowering.

Fine-tuning of flowering timing is crucial for plants to survive and leave offspring and depends on various endogenous and environmental factors. Here we report the identification of a vascular transcription factor, ANAC075, a putative regulator of VASCULAR-RELATED NAC-DOMAIN7 (VND7), as a negative regulator of flowering in Arabidopsis. Loss of function of ANAC075 causes the upregulation of floral integrator genes and early flowering under both long- and short-day conditions. ANAC075 promoter activity was detected in vascular tissues, including phloem. Previous reports suggested that ANAC075 is a transcriptional activator involved in the secondary cell wall formation, implying that the promotion of flowering time in anac075 mutants is caused by the disruption of flowering-time gene regulation in phloem and/or vascular tissue formation.

VP16 fusion induces the multiple-knockout phenotype of redundant transcriptional repressors partly by Med25-independent mechanisms in Arabidopsis.

Biological functions of only some plant transcriptional repressors are known owing to the lack of knockout lines or unclear phenotypes because of redundancy. Here we show that strong viral activation domain VP16 fusion to the transcriptional repressor FLOWERING LOCUS C reversed its function and caused a stronger phenotype than that of the multiple-knockout line of redundant genes, suggesting the potential of this technique to identify transcription factor function that cannot be detected in a single-knockout line. Loss-of-function of transcriptional coactivator Mediator25 did not affect VP16 activity despite their in vivo interaction, suggesting the existence of other key mechanism(s) in plants.

Chimeric repressor analysis identifies MYB87 as a possible regulator of morphogenesis via cell wall organization and remodeling in Arabidopsis.

Plant growth and development require proper cell wall organization but little is known about the transcription factors responsible for the regulation of gene expression involved in cell wall organization. Here we show, using Arabidopsis thaliana, that constitutive expression of the chimeric repressor for the MYB87 transcription factor causes suppression of longitudinal elongation, aberrant radial growth, and radially expanded or swollen cells in multiple organs. Microarray analysis revealed that plants expressing the chimeric repressor have altered expression of various cell wall related genes. MYB87 may therefore function as a regulator of genes affecting cell wall organization and remodeling. These findings improve our understanding of cell wall regulation and its roles in plant growth and development and also contribute information that may allow engineering of plant growth and architecture.

Overexpression of VOZ2 confers biotic stress tolerance but decreases abiotic stress resistance in Arabidopsis.

VOZ (vascular plant one zinc-finger protein) is a plant specific one-zinc finger type transcriptional activator, which is highly conserved through land plant evolution. We have previously shown that loss-of-function mutations in VOZ1 and VOZ2 showed increased cold and drought stress tolerances whereas decreased biotic stress resistance in Arabidopsis. Here, we demonstrate that transgenic plants overexpressing VOZ2 impairs freezing and drought stress tolerances but increases resistance to a fungal pathogen, Colletoricum higginsianum. Consistent with changes in the tolerance to biotic and abiotic stresses, the expression of marker genes for these stresses is significantly altered compared with those of the wild-type plant. These results indicate that a overexpression of VOZ2 confers biotic stress tolerance but impairs abiotic stress tolerances in Arabidopsis.

Vascular plant one-zinc-finger protein 1/2 transcription factors regulate abiotic and biotic stress responses in Arabidopsis.

Plants adapt to abiotic and biotic stresses by activating abscisic acid-mediated (ABA) abiotic stress-responsive and salicylic acid-(SA) or jasmonic acid-mediated (JA) biotic stress-responsive pathways, respectively. Although the abiotic stress-responsive pathway interacts antagonistically with the biotic stress-responsive pathways, the mechanisms that regulate these pathways remain largely unknown. In this study, we provide insight into the function of vascular plant one-zinc-finger proteins (VOZs) that modulate various stress responses in Arabidopsis. The expression of many stress-responsive genes was changed in the voz1voz2 double mutant under normal growth conditions. Consistent with altered stress-responsive gene expression, freezing- and drought-stress tolerances were increased in the voz1voz2 double mutant. In contrast, resistance to a fungal pathogen, Colletotrichum higginsianum, and to a bacterial pathogen, Pseudomonas syringae, was severely impaired. Thus, impairing VOZ function simultaneously conferred increased abiotic tolerance and biotic stress susceptibility. In a chilling stress condition, both the VOZ1 and VOZ2 mRNA expression levels and the VOZ2 protein level gradually decreased. VOZ2 degradation during cold exposure was completely inhibited by the addition of the 26S proteasome inhibitor, MG132, a finding that suggested that VOZ2 degradation is dependent on the ubiquitin/26S proteasome system. In voz1voz2, ABA-inducible transcription factor CBF4 expression was enhanced significantly even under normal growth conditions, despite an unchanged endogenous ABA content. A finding that suggested that VOZs negatively affect CBF4 expression in an ABA-independent manner. These results suggest that VOZs function as both negative and positive regulators of the abiotic and biotic stress-responsive pathways, and control Arabidopsis adaptation to various stress conditions.

A triantagonistic basic helix-loop-helix system regulates cell elongation in Arabidopsis.

In plants, basic helix-loop-helix (bHLH) transcription factors play important roles in the control of cell elongation. Two bHLH proteins, PACLOBTRAZOL RESISTANCE1 (PRE1) and Arabidopsis ILI1 binding bHLH1 (IBH1), antagonistically regulate cell elongation in response to brassinosteroid and gibberellin signaling, but the detailed molecular mechanisms by which these factors regulate cell elongation remain unclear. Here, we identify the bHLH transcriptional activators for cell elongation (ACEs) and demonstrate that PRE1, IBH1, and the ACEs constitute a triantagonistic bHLH system that competitively regulates cell elongation. In this system, the ACE bHLH transcription factors directly activate the expression of enzyme genes for cell elongation by interacting with their promoter regions. IBH1 negatively regulates cell elongation by interacting with the ACEs and thus interfering with their DNA binding. PRE1 interacts with IBH1 and counteracts the ability of IBH1 to affect ACEs. Therefore, PRE1 restores the transcriptional activity of ACEs, resulting in induction of cell elongation. The balance of triantagonistic bHLH proteins, ACEs, IBH1, and PRE1, might be important for determination of the size of plant cells. The expression of IBH1 and PRE1 is regulated by brassinosteroid, gibberellins, and developmental phase dependent factors, indicating that two phytohormones and phase-dependent signals are integrated by this triantagonistic bHLH system.

HSP90 functions in the circadian clock through stabilization of the client F-box protein ZEITLUPE.

The autoregulatory loops of the circadian clock consist of feedback regulation of transcription/translation circuits but also require finely coordinated cytoplasmic and nuclear proteostasis. Although protein degradation is important to establish steady-state levels, maturation into their active conformation also factors into protein homeostasis. HSP90 facilitates the maturation of a wide range of client proteins, and studies in metazoan clocks implicate HSP90 as an integrator of input or output. Here we show that the Arabidopsis circadian clock-associated F-box protein ZEITLUPE (ZTL) is a unique client for cytoplasmic HSP90. The HSP90-specific inhibitor geldanamycin and RNAi-mediated depletion of cytoplasmic HSP90 reduces levels of ZTL and lengthens circadian period, consistent with ztl loss-of-function alleles. Transient transfection of artificial microRNA targeting cytoplasmic HSP90 genes similarly lengthens period. Proteolytic targets of SCF(ZTL), TOC1 and PRR5, are stabilized in geldanamycin-treated seedlings, whereas the levels of closely related clock proteins, PRR3 and PRR7, are unchanged. An in vitro holdase assay, typically used to demonstrate chaperone activity, shows that ZTL can be effectively bound, and aggregation prevented, by HSP90. GIGANTEA, a unique stabilizer of ZTL, may act in the same pathway as HSP90, possibly linking these two proteins to a similar mechanism. Our findings establish maturation of ZTL by HSP90 as essential for proper function of the Arabidopsis circadian clock. Unlike metazoan systems, HSP90 functions here within the core oscillator. Additionally, F-box proteins as clients may place HSP90 in a unique and more central role in proteostasis.

Characterization of pseudo-response regulators in plants.

A small family of clock-regulated pseudo-response regulators (PRRs) plays a number of critical roles in the function of the plant circadian clock. The regulation of the PRRs is complex and entails both transcriptional and posttranslational regulation. PRR proteins engage in a number of important protein-protein interactions, some of which are modulated by modifications including phosphorylation. PRR stability is also tightly controlled. This chapter provides methods for studying both the PRR genes and their encoded proteins.

PRR5 regulates phosphorylation, nuclear import and subnuclear localization of TOC1 in the Arabidopsis circadian clock.

Many core oscillator components of the circadian clock are nuclear localized but how the phase and rate of their entry contribute to clock function is unknown. TOC1/PRR1, a pseudoresponse regulator (PRR) protein, is a central element in one of the feedback loops of the Arabidopsis clock, but how it functions is unknown. Both TOC1 and a closely related protein, PRR5, are nuclear localized, expressed in the same phase, and shorten period when deficient, but their molecular relationship is unclear. Here, we find that both proteins interact in vitro and in vivo through their conserved N-termini. TOC1-PRR5 oligomerization enhances TOC1 nuclear accumulation two-fold, most likely through enhanced nuclear import. In addition, PRR5 recruits TOC1 to large subnuclear foci and promotes phosphorylation of the TOC1 N-terminus. Our results show that nuclear TOC1 is essential for normal clock function and reveal a mechanism to enhance phase-specific TOC1 nuclear accumulation. Interestingly, this process of regulated nuclear import is reminiscent of similar oligomeric pairings in animal clock systems (e.g. timeless/period and clock/cycle), suggesting evolutionary convergence of a conserved mechanism across kingdoms.

SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis.

Endoreduplication involves a doubling of chromosomal DNA without corresponding cell division. In plants, many cell types transit from the mitotic cycle to the endoreduplication cycle or endocycle, and this transition is often coupled with the initiation of cell expansion and differentiation. Although a number of cell cycle regulators implicated in endocycle onset have been identified, it is still largely unknown how this transition is developmentally regulated at the whole organ level. Here, we report that a nuclear-localized SUMO E3 ligase, HIGH PLOIDY2 (HPY2), functions as a repressor of endocycle onset in Arabidopsis thaliana meristems. Loss of HPY2 results in a premature transition from the mitotic cycle to the endocycle, leading to severe dwarfism with defective meristems. HPY2 possesses an SP-RING domain characteristic of MMS21-type SUMO E3 ligases, and we show that the conserved residues within this domain are required for the in vivo and in vitro function of HPY2. HPY2 is predominantly expressed in proliferating cells of root meristems and it functions downstream of meristem patterning transcription factors PLETHORA1 (PLT1) and PLT2. These results establish that HPY2-mediated sumoylation modulates the cell cycle progression and meristem development in the PLT-dependent signaling pathway.