Almost all organisms coordinate some aspects of their biology through the diurnal cycle. Photosynthetic organisms, and plants especially, have established complex programs that coordinate physiological, metabolic and developmental processes with the changing light. The diurnal regulation of the underlying transcriptional processes is observed when groups of functionally related genes (gene modules) are expressed at a specific time of the day. However, studying the diurnal regulation of these gene modules in the plant kingdom was hampered by the large amount of data required for the analyses. To meet this need, we used gene expression data from 17 diurnal studies spanning the whole Archaeplastida kingdom (Plantae kingdom in the broad sense) to make an online diurnal database. We have equipped the database with tools that allow user-friendly cross-species comparisons of gene expression profiles, entire co-expression networks, co-expressed clusters (involved in specific biological processes), time-specific gene expression and others. We exemplify how these tools can be used by studying three important biological questions: (i) the evolution of cell division, (ii) the diurnal control of gene modules in algae and (iii) the conservation of diurnally controlled modules across species. The database is freely available at http://diurnal.plant.tools.

Angiosperms exhibit double fertilization, a process in which one of the sperm cells released from the pollen tube fertilizes the egg, while the other sperm cell fertilizes the central cell, giving rise to the embryo and endosperm, respectively. We have previously reported two polar nuclear fusion-defective double knockout mutants of Arabidopsis thaliana immunoglobulin binding protein (BiP), a molecular chaperone of the heat shock protein 70 (Hsp70) localized in the endoplasmic reticulum (ER), (bip1 bip2) and its partner ER-resident J-proteins, ERdj3A and P58IPK (erdj3a p58ipk). These mutants are defective in the fusion of outer nuclear membrane and exhibit characteristic seed developmental defects after fertilization with wild-type pollen, which are accompanied by aberrant endosperm nuclear proliferation. In this study, we used time-lapse live-cell imaging analysis to determine the cause of aberrant endosperm nuclear division in these mutant seeds. We found that the central cell of bip1 bip2 or erdj3a p58ipk double mutant female gametophytes was also defective in sperm nuclear fusion at fertilization. Sperm nuclear fusion was achieved after the onset of the first endosperm nuclear division. However, division of the condensed sperm nucleus resulted in aberrant endosperm nuclear divisions and delayed expression of paternally derived genes. By contrast, the other double knockout mutant, erdj3b p58ipk, which is defective in the fusion of inner membrane of polar nuclei but does not show aberrant endosperm nuclear proliferation, was not defective in sperm nuclear fusion at fertilization. We thus propose that premitotic sperm nuclear fusion in the central cell is critical for normal endosperm nuclear proliferation.

mRNA degradation is an important cellular mechanism involved in the control of gene expression. Several genome-wide profiling methods have been developed for detecting mRNA degradation in plants and animals. However, because many of these techniques use poly (A) mRNA for library preparation, degradation intermediates are often only detected near the 3′-ends of transcripts. Previously, we developed the Truncated RNA End Sequencing (TREseq) method using Arabidopsis thaliana, and demonstrated that this method ameliorates 3′-end bias. In analyses using TREseq, we observed G-rich sequences near the 5′-ends of degradation intermediates. However, this finding remained to be confirmed in other plant species. Hence, in this study, we conducted TREseq analyses in Lactuca sativa (lettuce), Oryza sativa (rice) and Rosa hybrida (rose). These species including A. thaliana were selected to encompass a diverse range in the angiosperm phylogeny. The results revealed similar sequence features near the 5′-ends of degradation intermediates, and involvement of translation process in all four species. In addition, homologous genes have similar efficiencies of mRNA degradation in different plants, suggesting that similar mechanisms of mRNA degradation are conserved across plant species. These strong sequence features were not observed in previous degradome analyses among different species in plants.

Plant cell wall polysaccharides, including xylan, glucomannan, xyloglucan and pectin, are often acetylated. Although a number of acetyltransferases responsible for the acetylation of some of these polysaccharides have been biochemically characterized, little is known about the source of acetyl donors and how acetyl donors are translocated into the Golgi, where these polysaccharides are synthesized. In this report, we investigated roles of ATP-citrate lyase (ACL) that generates cytosolic acetyl-CoA in cell wall polysaccharide acetylation and effects of simultaneous mutations of four Reduced Wall Acetylation (RWA) genes on acetyl-CoA transport into the Golgi in Arabidopsis thaliana. Expression analyses of genes involved in the generation of acetyl-CoA in different subcellular compartments showed that the expression of several ACL genes responsible for cytosolic acetyl-CoA synthesis was elevated in interfascicular fiber cells and induced by secondary wall-associated transcriptional activators. Simultaneous downregulation of the expression of ACL genes was demonstrated to result in a substantial decrease in the degree of xylan acetylation and a severe alteration in secondary wall structure in xylem vessels. In addition, the degree of acetylation of other cell wall polysaccharides, including glucomannan, xyloglucan and pectin, was also reduced. Moreover, Golgi-enriched membrane vesicles isolated from the rwa1/2/3/4 quadruple mutant were found to exhibit a drastic reduction in acetyl-CoA transport activity compared with the wild type. These findings indicate that cytosolic acetyl-CoA generated by ACL is essential for cell wall polysaccharide acetylation and RWAs are required for its transport from the cytosol into the Golgi.

In nature, photosynthetic organisms are exposed to highly dynamic environmental conditions where the excitation energy and electron flow in the photosynthetic apparatus need to be continuously modulated. Fluctuations in incident light are particularly challenging because they drive oversaturation of photosynthesis with consequent oxidative stress and photoinhibition. Plants and algae have evolved several mechanisms to modulate their photosynthetic machinery to cope with light dynamics, such as thermal dissipation of excited chlorophyll states (non-photochemical quenching, NPQ) and regulation of electron transport. The regulatory mechanisms involved in the response to light dynamics have adapted during evolution, and exploring biodiversity is a valuable strategy for expanding our understanding of their biological roles. In this work, we investigated the response to fluctuating light in Nannochloropsis gaditana, a eukaryotic microalga of the phylum Heterokonta originating from a secondary endosymbiotic event. Nannochloropsis gaditana is negatively affected by light fluctuations, leading to large reductions in growth and photosynthetic electron transport. Exposure to light fluctuations specifically damages photosystem I, likely because of the ineffective regulation of electron transport in this species. The role of NPQ, also assessed using a mutant strain specifically depleted of this response, was instead found to be minor, especially in responding to the fastest light fluctuations.

Nitric oxide (NO) is a crucial signaling molecule that conveys its bioactivity mainly through protein S-nitrosylation. This is a reversible post-translational modification (PTM) that may affect protein function. S-nitrosoglutathione (GSNO) is a cellular NO reservoir and NO donor in protein S-nitrosylation. The enzyme S-nitrosoglutathione reductase (GSNOR) degrades GSNO, thereby regulating indirectly signaling cascades associated with this PTM. Here, the two GSNORs of the legume Lotus japonicus, LjGSNOR1 and LjGSNOR2, have been functionally characterized. The LjGSNOR1 gene is very active in leaves and roots, whereas LjGSNOR2 is highly expressed in nodules. The enzyme activities are regulated in vitro by redox-based PTMs. Reducing conditions and hydrogen sulfide-mediated cysteine persulfidation induced both activities, whereas cysteine oxidation or glutathionylation inhibited them. Ljgsnor1 knockout mutants contained higher levels of S-nitrosothiols. Affinity chromatography and subsequent shotgun proteomics allowed us to identify 19 proteins that are differentially S-nitrosylated in the mutant and the wild-type. These include proteins involved in biotic stress, protein degradation, antioxidant protection and photosynthesis. We propose that, in the mutant plants, deregulated protein S-nitrosylation contributes to developmental alterations, such as growth inhibition, impaired nodulation and delayed flowering and fruiting. Our results highlight the importance of GSNOR function in legume biology.

Deschampsia antarctica is a Poaceae grass that has adapted to and colonized Antarctica. When D. antarctica plants were subjected to cold and dehydration stress both in the Antarctic field and in laboratory experiments, galactinol, a precursor of raffinose family oligosaccharides (RFOs) and raffinose were highly accumulated, which was accompanied by upregulation of galactinol synthase (GolS). The Poaceae monocots have a small family of GolS genes, which are divided into two distinct groups called types I and II. Type II GolSs are highly expanded in cold-adapted monocot plants. Transgenic rice plants, in which type II D. antarctica GolS2 (DaGolS2) and rice GolS2 (OsGolS2) were constitutively expressed, were markedly tolerant to cold and drought stress as compared to the wild-type rice plants. The RFO contents and GolS enzyme activities were higher in the DaGolS2- and OsGolS2-overexpressing progeny than in the wild-type plants under both normal and stress conditions. DaGolS2 and OsGolS2 overexpressors contained reduced levels of reactive oxygen species (ROS) relative to the wild-type plants after cold and drought treatments. Overall, these results suggest that Poaceae type II GolS2s play a conserved role in D. antarctica and rice in response to drought and cold stress by inducing the accumulation of RFO and decreasing ROS levels.

Medicago truncatula was proposed, about three decades ago, as a model legume to study the Rhizobium-legume symbiosis. It has now been adopted to study a wide range of biological questions, including various developmental processes (in particular root, symbiotic nodule and seed development), symbiotic (nitrogen-fixing and arbuscular mycorrhizal endosymbioses) and pathogenic interactions, as well as responses to abiotic stress. With a number of tools and resources set up in M. truncatula for omics, genetics and reverse genetics approaches, massive amounts of data have been produced, as well as four genome sequence releases. Many of these data were generated with heterogeneous tools, notably for transcriptomics studies, and are consequently difficult to integrate. This issue is addressed by the LeGOO (for Legume Graph-Oriented Organizer) knowledge base (https://www.legoo.org), which finds the correspondence between the multiple identifiers of the same gene. Furthermore, an important goal of LeGOO is to collect and represent biological information from peer-reviewed publications, whatever the technical approaches used to obtain this information. The information is modeled in a graph-oriented database, which enables flexible representation, with currently over 200,000 relations retrieved from 298 publications. LeGOO also provides the user with mining tools, including links to the Mt5.0 genome browser and associated information (on gene functional annotation, expression, methylome, natural diversity and available insertion mutants), as well as tools to navigate through different model species. LeGOO is, therefore, an innovative database that will be useful to the Medicago and legume community to better exploit the wealth of data produced on this model species.

Cyperus esculentus is probably the only plant that is known to accumulate large amounts of oil in its tubers. However, the underlying metabolic mechanism and regulatory factors involved in oil synthesis of tubers are still largely unclear. In this study, one gene encoding type I diacylglycerol acyltransferase (DGAT) (CeDGAT1) and two genes encoding type II DGAT (CeDGAT2a and CeDGAT2b) from C. esculentus were identified and functionally analyzed. All three DGAT genes were found to be expressed in tuber, root and leaf tissues. CeDGAT1 is highly expressed in roots and leaves, whereas CeDGAT2b is dominantly expressed in tubers. Furthermore, the temporal expression pattern of CeDGAT2b is well coordinated with the oil accumulation in developing tubers. When each CeDGAT was heterologously expressed in triacylglycerol (TAG)-deficient mutant of Saccharomyces cerevisiae, Arabidopsis thaliana wild type or its TAG1 mutant with AtDGAT1 disruption, only CeDGAT2b showed the ability to restore TAG biosynthesis with lipid body formation in yeast mutant, enhance seed oil production of Arabidopsis wild type and rescue multiple seed phenotypes of TAG1 mutant. In addition, CeDGAT2b was shown to have a substrate preference for unsaturated fatty acids toward TAG synthesis. Taken together, our results indicated that CeDGAT2b from C. esculentus is an actively functional protein and is most likely the major contributor to tuber oil biosynthesis containing common fatty acids, in contrast to oil-rich seeds and fruits where DGAT1 plays a more central role than DGAT2 in oil production accumulating normal fatty acids, whereas DGAT2 is a primary regulator for oil synthesis rich in unusual fatty acids.

As an important environment factor, light affects plant growth and development throughout life. B-BOX (BBX) proteins play key roles in the regulation of light signaling. Although the multiple roles of BBX proteins have been extensively studied in Arabidopsis, the research in apple is much less extensive. In this study, we systematically characterized the negative role of an apple BBX protein MdBBX37 in light signaling, including inhibiting anthocyanin biosynthesis and promoting hypocotyl elongation. We found that MdBBX37 interacted with MdMYB1 and MdMYB9, two key positive regulators of anthocyanin biosynthesis, and inhibited the binding of those two proteins to their target genes and, therefore, negatively regulated anthocyanin biosynthesis. In addition, MdBBX37 directly bound to the promoter of MdHY5, a positive regulator of light signaling, and suppressed its expression, and thus relieved MdHY5-mediated hypocotyl inhibition. Taken together, our investigations suggest that MdBBX37 is a negative regulator of light signaling in apple. Our study will provide reference for further study on the functions of BBX proteins in apple.

A flower is an essential organ for sexual reproduction in flowering plants, which has been extensively studied in model plants. In this study, we used transcriptomic, small RNA and degradome analyses to characterize key microRNAs (miRNAs) and their targets in floral organs of moso bamboo. In total, we identified 13,051 differentially expressed genes and 109 known miRNAs from 26 miRNA families. We aligned the miRNAs to known miRNA databases and revealed some conserved as well as novel miRNAs. Sixteen conserved miRNAs were specifically and highly expressed in stamens, including miRNA159 and miRNA166. In situ hybridization shows that miRNA159 plays a key role in the regulation of stamen development, and the expression levels of its targets PheMYB98 and PheMYB42 were low. Furthermore, Phe-MIRNA159 partially recovers phenotypes of mir159ab double mutant. Overexpression of Phe-MIR159 could cause failure in anther dehisce, and the mature pollens could not be dispersed and further reduce fertility in Arabidopsis. Semi-thin section result shows that anther endothelial layer of Phe-MIRNA159 overexpressing lines is lack of secondary thickening, resulting in limited force for anther opening. Phe-miR159 may regulate the expression of genes related to secondary thickening through negative regulation of AtMYB33, affecting the anther dehiscence. Taken together, this study provides insights regarding molecular networks underlying floral organs development of moso bamboo.

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic RNA surveillance mechanism that degrades aberrant mRNAs comprising a premature translation termination codon. The adenosine triphosphate (ATP)-dependent RNA helicase up-frameshift 1 (UPF1) is a major NMD factor in all studied organisms; however, the complexity of this mechanism has not been fully characterized in plants. To identify plant NMD factors, we analyzed UPF1-interacting proteins using tandem affinity purification coupled to mass spectrometry. Canonical members of the NMD pathway were found along with numerous NMD candidate factors, including conserved DEA(D/H)-box RNA helicase homologs of human DDX3, DDX5 and DDX6, translation initiation factors, ribosomal proteins and transport factors. Our functional studies revealed that depletion of DDX3 helicases enhances the accumulation of NMD target reporter mRNAs but does not result in increased protein levels. In contrast, silencing of DDX6 group leads to decreased accumulation of the NMD substrate. The inhibitory effect of DDX6-like helicases on NMD was confirmed by transient overexpression of RH12 helicase. These results indicate that DDX3 and DDX6 helicases in plants have a direct and opposing contribution to NMD and act as functional NMD factors.

Heat stress poses a major threat to plant productivity and crop yields. The induction of heat shock proteins (HSPs) by heat shock factors is a principal defense response of plants exposed to heat stress. In this study, we identified and analyzed the heat stress-induced Whirly1 (SlWHY1) gene in tomato (Solanum lycopersicum). We generated various SlWHY1-overexpressing (OE) and SlWHY1-RNA interference (RNAi) lines to investigate the role of WHIRLY1 in thermotolerance. Compared with the wild type (WT), the OE lines showed less wilting, as reflected by their increased membrane stability and soluble sugar content and reduced reactive oxygen species (ROS) accumulation under heat stress. By contrast, RNAi lines with inhibited SlWHY1 expression showed the opposite phenotype and corresponding physiological indices under heat stress. The heat-induced gene SlHSP21.5A, encoding an endoplasmic reticulum-localized HSP, was upregulated in the OE lines and downregulated in the RNAi lines compared with the WT. RNAi-mediated inhibition of SlHSP21.5A expression also resulted in reduced membrane stability and soluble sugar content and increased ROS accumulation under heat stress compared with the WT. SlWHY1 binds to the elicitor response element-like element in the promoter of SlHSP21.5A to activate its transcription. These findings suggest that SlWHY1 promotes thermotolerance in tomato by regulating SlHSP21.5A expression.

Microalgae such as Chlamydomonas reinhardtii accumulate triacylglycerol (TAG), which is a potential source of biofuels, under stress conditions such as nitrogen deprivation, whereas Chlamydomonas debaryana NIES-2212 has previously been identified and characterized as one of the rare species of Chlamydomonas, which massively accumulates TAG in the stationary phase without external stress. As the high density of the cells in the stationary phase was supposed to act as a trigger for the accumulation of TAG in C. debaryana, in this study, C. debaryana was encapsulated in a Ca2+-alginate gel for the culture with high cell density. We discovered that the growth of the encapsulated cells resulted in the formation of spherical palmelloid colonies with high cell density, where daughter cells with truncated flagella remained wrapped within the mother cell walls. Interestingly, gel encapsulation markedly promoted proliferation of C. debaryana cells, and the encapsulated cells reached the stationary phase earlier than that of the free-living cells. Gel encapsulation also enhanced TAG accumulation. Gene expression analysis revealed that two genes of acyltransferases, DGAT1 and DGTT3, were upregulated in the stationary phase of free-living C. debaryana. In addition, the gene expression of these acyltransferases increased earlier in the encapsulated cells than that in the free-living cells. The enhanced production of TAG by alginate gel encapsulation was not found in C. reinhardtii which is known to use a different repertoire of acyltransferases in lipid accumulation.

Serine/arginine-rich (SR) proteins have an essential role in the splicing of pre-messenger RNA (pre-mRNA) in eukaryote. Pre-mRNA with introns can be alternatively spliced to generate multiple transcripts, thereby increasing adaptation to the external stress conditions in planta. However, pre-mRNA of SR proteins can also be alternatively spliced in different plant tissues and in response to diverse stress treatments, indicating that SR proteins might be involved in regulating plant development and adaptation to environmental changes. We identified and named 18 SR proteins in cassava and systematically studied their splicing and transcriptional changes under tissue-specific and abiotic stress conditions. Fifteen out of 18 SR genes showed alternative splicing in the tissues. 45 transcripts were found from 18 SR genes under normal conditions, whereas 55 transcripts were identified, and 21 transcripts were alternate spliced in some SR genes under salt stress, suggesting that SR proteins might participate in the plant adaptation to salt stress. We then found that overexpression of MeSR34 in Arabidopsis enhanced the tolerance to salt stress through maintaining reactive oxygen species homeostasis and increasing the expression of calcineurin B-like proteins (CBL)–CBL-interacting protein kinases and osmotic stress-related genes. Therefore, our findings highlight the critical role of cassava SR proteins as regulators of RNA splicing and salt tolerance in planta.

It is well known that far-red light (FR; >700 nm) drives PSI photochemistry, but its effect on photosynthetic performance has received little attention. In this study, the effects of the addition of FR to red fluctuating light (FL) have on photosynthesis were examined in the leaves of Arabidopsis thaliana. Light-activated leaves were illuminated with FL [alternating high light/low light (HL/LL) at 800/30 μmol m−2 s−1] for 10–15 min without or with FR at intensities that reflected natural conditions. The CO2 assimilation rates upon the transition from HL to LL were significantly greater with FR than without FR. The enhancement of photosynthesis by FR was small under the steady-state conditions and in the HL phases of FL. Proton conductivity through the thylakoid membrane (gH+) in the LL phases of FL, estimated from the dark relaxation kinetics of the electrochromic absorbance shift, was greater with FR than without FR. The relaxation of non-photochemical quenching (NPQ) in the PSII antenna system and the increase in PSII photochemistry in the LL phases accelerated in the presence of FR. Similar FR-effects in FL were confirmed in typical sun and shade plants. On the basis of these results, we concluded that FR exerted beneficial effects on photosynthesis in FL by exciting PSI and accelerating NPQ relaxation and PSII-yield increase. This was probably because of the increased gH+, which would reflect faster ΔpH dissipation and ATP synthesis.

Frost stress severely affects agriculture and agroforestry worldwide. Although many studies about frost hardening and resistance have been published, most of them focused on the aboveground organs and only a minority specifically targets the roots. However, roots and aboveground tissues have different physiologies and stress response mechanisms. Climate models predict an increase in the magnitude and frequency of late-frost events, which, together with an observed loss of soil insulation, will greatly decrease plant primary production due to damage at the root level. Molecular and metabolic responses inducing root cold hardiness are complex. They involve a variety of processes related to modifications in cell wall composition, maintenance of the cellular homeostasis and the synthesis of primary and secondary metabolites. After a summary of the current climatic models, this review details the specificity of freezing stress at the root level and explores the strategies roots developed to cope with freezing stress. We then describe the level to which roots can be frost hardy, depending on their age, size category and species. After that, we compare the environmental signals inducing cold acclimation and frost hardening in the roots and aboveground organs. Subsequently, we discuss how roots sense cold at a cellular level and briefly describe the following signal transduction pathway, which leads to molecular and metabolic responses associated with frost hardening. Finally, the current options available to increase root frost tolerance are explored and promising lines of future research are discussed.

Flowering plants reproduce via a complex double fertilization process. Two female gamete cells, the egg cell and the central cell, reside in the ovule. Once fertilized by a sperm cell, they generate the seed embryo and endosperm, respectively (Kawashima and Berger 2011). Unlike the haploid egg cell, the central cell is a homodiploid: two female gametophytic nuclei, known as the polar nuclei, fuse with each other and establish the diploid central cell nucleus. The nuclear envelope consists of inner and outer membranes, and these must combine to complete nuclear fusion. After the male and female gametes fuse (in plasmogamy), the male nucleus migrates toward the female nucleus to undergo gamete nuclear fusion or karyogamy (Fatema et al. 2019). In the model plant Arabidopsis thaliana, several genes have been identified that play a role in fusion of the polar nuclei and mutations in these genes show endosperm developmental defects (Erbasol Serbes et al. 2019); however, until now, it has remained unclear whether the aberrant endosperm phenotype was a direct result of this incorrect fusion. Using a time-lapse live-cell imaging approach, Maruyama et al. (2019) utilized mutants defective in each membrane fusion factor to carefully assess the significance of polar nuclear fusion for the initiation of endosperm development (Fig. 1).

Plant and Cell Physiology,https://doi.org/10.1093/pcp/pcz169Published: 28 August 2019

Tomato plants (Solanum lycopersicum) contain steroidal glycoalkaloid α-tomatine, which functions as a chemical barrier to pathogens and predators. α-Tomatine accumulates in all tissues and at particularly high levels in leaves and immature green fruits. The compound is toxic and causes a bitter taste, but its presence decreases through metabolic conversion to nontoxic esculeoside A during fruit ripening. This study identifies the gene encoding a 23-hydroxylase of α-tomatine, which is a key to this process. Some 2-oxoglutarate-dependent dioxygenases were selected as candidates for the metabolic enzyme, and Solyc02g062460, designated Sl23DOX, was found to encode α-tomatine 23-hydroxylase. Biochemical analysis of the recombinant Sl23DOX protein demonstrated that it catalyzes the 23-hydroxylation of α-tomatine and the product spontaneously isomerizes to neorickiioside B, which is an intermediate in α-tomatine metabolism that appears during ripening. Leaves of transgenic tomato plants overexpressing Sl23DOX accumulated not only neorickiioside B but also another intermediate, lycoperoside C (23-O-acetylated neorickiioside B). Furthermore, the ripe fruits of Sl23DOX-silenced transgenic tomato plants contained lower levels of esculeoside A but substantially accumulated α-tomatine. Thus, Sl23DOX functions as α-tomatine 23-hydroxylase during the metabolic processing of toxic α-tomatine in tomato fruit ripening and is a key enzyme in the domestication of cultivated tomatoes.

Late embryogenesis abundant (LEA) proteins comprise a large family that play important roles in the regulation of abiotic stresses. No in-depth analysis of LEA genes has been performed in grapevine. In this study, we analyzed a total of 52 putative LEA genes in grapevine at genomic and transcriptomic levels, compiled expression profiles of four selected VamLEA genes under cold and osmotic stress and studied potential function of VamDHN3 gene in grapevine callus. The 52 LEA proteins can be classified into seven phylogenetic groups. RNA-seq and qRT-PCR results demonstrated that a total of 16 and 23 VamLEA genes were upregulated under cold and osmotic stress. Overexpression of VamDHN3 enhances the stability of cell membrane in grapevine callus, suggesting VamDHN3 is involved in osmotic regulation. These results formed fundamental knowledge for further analysis of biological roles of LEA genes in grapevine’s adaption to abiotic stresses.

Stem cells undergo cell division and differentiation to ensure organized tissue development. Because plant cells are immobile, plant stem cells ought to decide their cell fate prior to differentiation, to locate specialized cells in the correct position. In this study, based on a chemical screen we isolated a novel secondary cell wall indicator BF-170, which binds to lignin and can be used to image in vitro and in situ xylem development. Use of BF-170 to observe the vascular differentiation pattern in the in vitro vascular cell induction system, VISUAL revealed that adaxial mesophyll cells of cotyledons predominantly generate ectopic xylem cells. Moreover, phloem cells are abundantly produced on the abaxial layer, suggesting the involvement of leaf adaxial-abaxial polarity in determining vascular cell fate. Analysis of abaxial polarity mutants highlighted the role of YAB3, an abaxial cell fate regulator, in suppressing xylem and promoting phloem differentiation on the abaxial domains in VISUAL. Further YABBY family genes also affected in vivo vascular development during the secondary growth. Our results denoted the possibility that such mediators of spatial information contribute to correctly determine the cell fate of vascular stem cells, to conserve the vascular pattern of land plants.

Soil salinity, a prevalent abiotic stress, causes enormous losses in global crop yields annually. Previous studies have shown that salt stress-induced reprogramming of gene expression contributes to the survival of plants under this stress. However, mechanisms regulating gene expression in response to salt stress at the posttranscriptional level are not well understood. Here, we show that salt stress increases the level of Signal Responsive 1 (SR1) mRNA, a member of signal-responsive Ca2+/calmodulin-regulated transcription factors, by enhancing its stability. We present multiple lines of evidence indicating that reactive oxygen species generated by NADPH oxidase activity mediate salt-induced SR1 transcript stability. Using mutants impaired in either nonsense-mediated decay, XRN4 or mRNA decapping pathways, we show that neither the NMD pathway, XRN4 nor the decapping of SR1 mRNA is required for its decay. We analyzed the salt-induced accumulation of eight truncated versions of the SR1 coding region (∼3 kb) in the sr1 mutant background. This analysis identified a 500 nts region at the 3’ end of the SR1 coding region to be required for the salt-induced stability of SR1 mRNA. Potential mechanisms by which this region confers SR1 transcript stability in response to salt are discussed.

Autopolyploids often show growth advantages over their diploid progenitors because of their increased photosynthetic activity; however, the underlying molecular basis of such mechanism remains elusive. Here, we aimed to characterize autotetraploid pak choi (Brassica rapa ssp. chinensis) at the physiological, cellular, and molecular levels. Autotetraploid pak choi has thicker leaves than its diploid counterparts, with relatively larger intercellular spaces and cell size and greater grana thylakoid height. Photosynthetic data showed that the relative electron transport rate (rETR) was markedly higher in autotetraploid than in diploid pak choi. Transcriptomic data revealed that the expressions of genes involved in ‘photosynthesis’ biological process and ‘thylakoids’ cellular component were mainly regulated in autotetraploids. Overall, our findings suggested that the increased rETR in the thylakoids contributed to the increased photosynthetic capacity of autotetraploid leaves. Furthermore, we found that the enhanced rETR is associated with increased BrPetC expression, which is likely altered by histone modification. The ectopic expression of BrPetC in Arabidopsis thaliana led to increased rETR and biomass, which were decreased in BrPetC-silenced pak choi. Autotetraploid pak choi also show altered hormone levels, which was likely responsible for the increased drought resistance and impaired powdery mildew resistance of this lineage. Our findings further our understanding on how autotetraploidy provides growth advantages to plants.

Acyl-CoA-binding proteins (ACBPs) are involved in binding and trafficking acyl-CoA esters in eukaryotic cells. ACBPs contain a well-conserved acyl-CoA-binding domain (ACBD). Their various functions have been characterized in the model plant Arabidopsis and, to a lesser extent, in rice. In this study, genome-wide detection and expression analysis of ACBPs were performed on Elaeis guineensis (oil palm), the most important oil crop in the world. Seven E. guineensis ACBPs were identified and classified into four groups according to their deduced amino acid domain organization. Phylogenetic analysis showed conservation of this family with other higher plants. All seven EgACBPs were expressed in most tissues while their differential expression suggests various functions in specific tissues. For example, EgACBP3 had high expression in inflorescences and stalks while ACBP1 showed strong expression in leaves. Because of the importance of E. guineensis as an oil crop, expression of EgACBPs was specifically examined during fruit development. EgACBP3 showed high expression throughout mesocarp development, while EgACBP1 had enhanced expression during rapid oil synthesis. In endosperm, both EgACBP1 and EgACBP3 exhibited increased expression during seed development. These results provide important information for further investigations on the biological functions of EgACBPs in various tissues and, in particular, their roles in oil synthesis.

Brassicaceae and closely related species develop unique endoplasmic reticulum (ER)-derived structures called ER bodies, which accumulate β-glucosidases/myrosinases that are involved in chemical defense. There are two different types of ER bodies: ER bodies constitutively present in seedlings (cER bodies) and ER bodies in rosette leaves induced by treatment with the wounding hormone jasmonate (iER bodies). Here, we show that At-ɑ whole genome duplication (WGD) generated the paralogous genes NAI2 and TSA1, which consequently drive differentiation of cER bodies and iER bodies in Brassicaceae plants. In Arabidopsis, NAI2 is expressed in seedlings where cER bodies are formed, whereas TSA1 is expressed in jasmonate treated leaves where iER bodies are formed. We found that the expression of NAI2 in seedlings and the jasmonate inducibility of TSA1 are conserved across other Brassicaceae plants. The accumulation of NAI2 transcripts in Arabidopsis seedlings is dependent on the transcription factor NAI1, whereas the jasmonate induction of TSA1 in rosette leaves is dependent on MYC2, MYC3, and MYC4. We discovered regions of microsynteny, including the NAI2/TSA1 genes, but the promoter regions are differentiated between TSA1 and NAI2 genes in Brassicaceae. This suggests that the divergence of function between NAI2 and TSA1 occurred immediately after WGD in ancestral Brassicaceae plants to differentiate the formation of iER and cER bodies. Our findings indicate that At-ɑ WGD enabled diversification of defense strategies, which may have contributed to the massive diversification of Brassicaceae plants.

Development of pollen, the male gametophyte of flowering plants, is tightly controlled by dynamic changes in gene expression. Recent research to clarify the molecular aspects of pollen development has revealed the involvement of several transcription factors in the induction of gene expression. However, limited information is available about the factors involved in the negative regulation of gene expression to eliminate unnecessary transcripts during pollen development. In this study, we revealed that AtNOT1 is an essential protein for proper pollen development and germination capacity. AtNOT1 is a scaffold protein of the AtCCR4-NOT complex, which includes multiple components related to mRNA turnover control in Arabidopsis. Phenotypic analysis using atnot1 heterozygote mutant pollen showed that the mature mutant pollen failed to germinate, and also revealed abnormal localization of nuclei and a specific protein at the tri-cellular pollen stage. Furthermore, transcriptome analysis of atnot1 heterozygote mutant pollen demonstrated that the down-regulation of a large number of transcripts, along with the up-regulation of specific transcripts required for pollen tube germination by AtNOT1 during late microgametogenesis, is important for proper pollen development and germination. Overall, our findings provide new insights into the negative regulation of gene expression during pollen development, by demonstrating the severely defective phonotype of atnot1 heterozygote mutant pollen.

The presence of small tooth-like indentations, or serrations, characterizes leaf margins of Arabidopsis thaliana plants. The NAC family member CUC2, which undergoes post-transcriptional gene silencing by three micro RNA genes (MIR164A, B, and C), controls the extension of leaf serration. Here we analyzed the role of AtHB1, a transcription factor (TF) belonging to the homeodomain-leucine zipper subfamily I, in shaping leaf margins. Using mutants with an impaired silencing pathway as background, we obtained transgenic plants expressing AtHB1 over 100 times compared to controls. These plants presented an atypical developmental phenotype characterized by leaves with deep serration. Transcript measurements revealed that CUC2 expression was induced in plants overexpressing AtHB1 and repressed in athb1 mutants, indicating a positive regulation exerted by this TF. Moreover, molecular analyses of AtHB1 overexpressing and mutant plants revealed that AtHB1 represses MIR164 transcription. We found that overexpression of MIR164B was able to reverse the serration phenotype of plants overexpressing AtHB1. Finally, chromatin immunoprecipitation assays revealed that AtHB1 was able to bind in vivo the promoter regions of all three MIR164 encoding loci. Altogether, our results indicate that AtHB1 directly represses MIR164 expression to enhance leaf serration by increasing CUC2 levels.

Iron (Fe) deficiency limits the yield of fruit trees. When subjected to Fe-deficiency, H+ secretion increases in the rhizosphere of dicotyledonous plants and pH decreases. This leads to the acidification of the soil and promotes Fe3+ to Fe2+ conversion, which plants can better uptake. This study investigated the relationship between two inhibitory transcription factors (ethylene response factors MbERF4 and MbERF72) and the H+-ATPase gene MbHA2. Two species of apple woody plants were studied: the Fe-inefficient Malus baccata and the Fe-efficient Malus xiaojinensis. Yeast one-hybrid and electrophoretic mobility shift assays showed that both MbERF4 and MbERF72 bind to the GCC cassette (AGCCGCC) of the MbHA2 promoter. Moreover, yeast two-hybrid and bimolecular fluorescence complementation assays showed that MbERF4 interacts with MbERF72. Furthermore, β-glucuronidase and luciferase reporter assays showed that the MbERF4- and MbERF72-induced repression of MbHA2 expression is synergistic. Virus-induced gene silencing of MbERF4 or MbERF72 increased MbHA2 expression, and thus lowered the rhizosphere pH in M. baccata. Consequently, the high expressions of MbERF4 and MbERF72 induced by Fe-deficiency contributed to the Fe sensitivity of M. baccata. Moreover, the low expressions of MxERF4 and MxERF72 contributed to the Fe-deficiency tolerance of M. xiaojinensis via different binding conditions to the HA2 promoter. In summary, this study identified the relationship of two inhibitory transcription factors with the H+-ATPase gene and proposed a model in which ERF4 and ERF72 affect the rhizosphere pH in response to Fe-deficiency.

The involvement of small auxin-up RNA proteins (SAURs) in leaf senescence has been more and more acknowledged, but the detailed mechanisms remain unclear. In the present study, we performed yeast two-hybrid assays (Y2Hs) and identified SAUR49 as an interactor of SSPP, which is a PP2C protein phosphatase that negatively regulates Arabidopsis leaf senescence by suppressing the leucine-rich repeat receptor-like protein kinase (LRR-RLK) SARK, as reported previously by our group. The interaction between SAUR49 and SSPP was further confirmed in planta. Functional characterization revealed that SAUR49 is a positive regulator of leaf senescence. The accumulation level of SAUR49 protein increased during natural leaf senescence in Arabidopsis. The transcript level of SAUR49 was upregulated during SARK-induced premature leaf senescence but downregulated during SSPP-mediated delayed leaf senescence. Overexpression of SAUR49 significantly accelerated both natural and dark-induced leaf senescence in Arabidopsis. More importantly, SAUR49 overexpression completely reversed SSPP-induced delayed leaf senescence. In addition, overexpression of SAUR49 reversed the decreased plasma membrane (PM) H+-ATPase activity mediated by SSPP. Taken together, the results showed that SAUR49 functions in accelerating the leaf senescence process via the activation of SARK-mediated leaf senescence signalling by suppressing SSPP. We further identified four other SSPP-interacting SAURs, SAUR30, SAUR39, SAUR41 and SAUR72, that may act redundantly with SAUR49 in regulating leaf senescence. All these observations indicated that certain members of the SAUR family may serve as an important hub that integrates various hormonal and environmental signals with senescence signals in Arabidopsis.

To optimize growth and development, plants monitor photosynthetic activities and appropriately regulate various cellular processes. However, signaling mechanisms that coordinate plant growth with photosynthesis remain poorly understood. To identify factors that are involved in signaling related to photosynthetic stimuli, we performed a phosphoproteomic analysis with Marchantia polymorpha, an extant bryophyte species in the basal lineage of land plants. Among proteins whose phosphorylation status changed differentially between dark-treated plants and those after light irradiation, but failed to do so in the presence of a photosynthesis inhibitor, we identified a B4-group Raf-like kinase, named PHOTOSYNTHESIS-RELATED RAF (MpPRAF). Biochemical analyses confirmed photosynthesis-activity-dependent changes in the phosphorylation status of MpPRAF. Mutations in the MpPRAF gene resulted in growth retardation. Measurement of carbohydrates demonstrated both hyper-accumulation of starch and reduction of sucrose in Mppraf mutants. Neither inhibition of starch synthesis nor exogenous supply of sucrose alleviated the growth defect, suggesting serious impairment of Mppraf mutants in both the synthesis of sucrose and the repression of its catabolism. As a result of the compromised photosynthate metabolism, photosynthetic electron transport was down-regulated in Mppraf mutants. A mutated MpPRAF with a common amino-acid substitution for inactivating kinase activity was unable to rescue the Mppraf mutant defects. Our results provide evidence that MpPRAF is a photosynthesis signaling kinase that regulates sucrose metabolism.

Appropriate cell cycle regulation is crucial for achieving coordinated development and cell differentiation in multicellular organisms. In Arabidopsis, endoreduplication is often observed in terminally differentiated cells, and several reports have shown its molecular mechanisms. Auxin is a key factor for the mode transition from mitotic cell cycle to endocycle; however, it remains unclear if and how auxin maintains the endocycle mode. Here, we reanalyzed root single-cell transcriptome data and reconstructed cell cycle trajectories of the mitotic cell cycle and endocycle. With progression of the endocycle, genes involved in auxin synthesis, influx, and efflux were induced at the specific cell phase, suggesting that auxin concentration fluctuated dynamically. Such induction of auxin-related genes was not observed in the mitotic cell cycle, suggesting that the auxin fluctuation plays some roles in maintaining the endocycle stage. In addition, the expression level of CYCB1;1, which is required for cell division in the M phase, coincided with the expected amount of auxin and cell division. Our analysis also provided a set of genes expressed in specific phases of the cell cycle. Taking these findings together, reconstruction of single-cell transcriptome data enables us to identify properties of the cell cycle more accurately.

Taxane diterpenes are secondary metabolites with an important pharmacological role in the treatment of cancer. Taxus spp. biofactories have been used for taxane production, but the lack of knowledge about the taxane biosynthetic pathway and its molecular regulation hinders their optimal function. The difficulties in introducing foreign genes in Taxus spp. genomes hinders the study of the molecular mechanisms involved in taxane production and a new approach is required to overcome them. In this work, a reliable, simple and fast method to obtain Taxus x media protoplasts was developed, allowing their manipulation in downstream assays for the study of physiological changes in Taxus spp. cells. Using this method, Taxus protoplasts were transiently transfected for the first time, corroborating their suitability for transfection assays and the study of specific physiological responses. The two assayed transcription factors (BIS2 and TSAR2) had a positive effect on the expression of several taxane-related genes, suggesting their potential use for the improvement of taxane yields. Furthermore, the results indicate that the developed method is suitable for obtaining Taxus x media protoplasts for transfection with the aim of unravelling regulatory mechanisms in taxane production.

Reversible protein phosphorylation orchestrated by protein kinases and phosphatases is a major regulatory event in plants and animals. The SnRK2 subfamily consists of plant-specific protein kinases in the Ser/Thr protein kinase superfamily. Early observations indicated that SnRK2s are mainly involved in response to abiotic stress. Recent evidence shows that SnRK2s are multifarious players in a variety of biological processes. Here, we summarize the considerable knowledge of SnRK2s, including evolution, classification, biological functions, and regulatory mechanisms at the epigenetic, post-transcriptional, and post-translation levels.

Arabidopsis (Arabidopsis thaliana) 12-oxophytodienoic acid reductase isoform 3 (OPR3) is involved in the synthesis of jasmonic acid by reducing the α,β-unsaturated double bond of the cyclopentenone moiety in 12-oxo-phytodienoic acid (12-OPDA). Recent research revealed that jasmonic acid synthesis is not strictly dependent on the peroxisomal OPR3. The ability of OPR3 to reduce trinitrotoluene suggests that the old yellow enzyme homologue OPR3 has additional functions. Here we demonstrate that OPR3 catalyzes the reduction of a wide spectrum of electrophilic species that share a reactivity towards the major redox buffers glutathione (GSH) and ascorbate (ASC). Furthermore, we demonstrate that 12-OPDA reacts with ascorbate to form an ASC-12-OPDA adduct, but in addition OPR3 has the ability to regenerate ASC from monodehydroascorbate (MDHA). The presented data characterize OPR3 as a bifunctional enzyme with NADPH-dependent α,β-ketoalkene double bond reductase and monodehydroascorbate reductase activities (MDHAR). opr3 mutants exhibited a slightly less reduced ASC pool in leaves in line with the MDHAR activity of OPR3 in vitro. These functions link redox homeostasis as mediated by ASC and GSH with OPR3 activity and metabolism of reactive electrophilic species (RES).

Energy cane is a bioenergy crop with an outstanding ability to bud sprouting and increasing yield in ratoon cycles even in marginal lands. Bud fate control is key to biomass production and crop profits due to vegetative propagation and tiller dependency, as well as phenotype plasticity to withstand harsh environmental conditions. During the establishment stage (plant-cane cycle), energy cane has a tendency for low root : shoot ratio, which might hamper the ability to cope with stress. Auxin is known to modulate bud sprouting and stimulate rooting in sugarcane. Hence, we treated a slow and a fast bud-sprouting energy cane cultivars with auxin or controls (with and without water soaking) for 6 h prior to planting and evaluate plant growth parameters and metabolic profiling using two techniques (GC-TOF-MS and NMR) to characterize the effect and identify metabolite markers associated with bud inhibition and outgrowth. Auxin inhibited bud burst and promote rooting in setts changing the root : shoot ratio of plantlets. Metabolome allowed the identification of lactate, succinate and aspartate-family amino acids as involved in bud fate control through the potential modulation of oxygen and energy status. Investigating environmental and biochemical factors that regulate bud fate can be incremental to other monocot species. Our study provides new insights into bud quiescence and outgrowth in cane hybrids, with the potential to leverage our understanding of yield-related traits, crop establishment and adaptation to global climate change.

WRKY is one of the largest transcription factor families in plants, and plays important roles in the regulation of developmental and physiological processes. To date, the WRKY gene family has not been identified in Saccharum species because of its complex polyploid genome. In this study, a total of 294 sequences for 154 SsWRKY genes were identified in the polyploid Saccharum spontaneum genome and then named on the basis of their chromosome locations, including 13 (8.4%) genes with four alleles, 29 (18.8%) with three alleles and 41 (26.6%) with two alleles. Among them, 73.8% and 16.0% of the SsWRKY genes originated from segmental duplications and tandem duplications, respectively. The WRKY members exhibited conserved gene structures and amino acid sequences among the allelic haplotypes which were accompanied by variations in intron sizes. Phylogenetic and collinearity analysis revealed that 27 SsWRKYs originated after the split of sorghum and Saccharum, resulting in a significantly higher number of WRKYs in sugarcane than the proximal diploid species sorghum. The analysis of RNA-seq data revealed that SsWRKYs expression profiles in 46 different samples including different developmental stages revealed distinct temporal and spatial patterns with 52 genes expressed in all tissues, 4 genes not expressed in any tissues, and 21 SsWRKY genes likely to be involved in photosynthesis. The comprehensive analysis of SsWRKYs expression will provide an important and valuable foundation for further investigation of the regulatory mechanisms of WRKYs in physiological roles in sugarcane Saccharum spontaneum.

Auxin is the first discovered plant hormone and is essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the main auxin and plays pivotal roles in intercellular communication through polar auxin transport. Phenylacetic acid (PAA) is another natural auxin that does not show polar movement. Although a wide range of species have been shown to produce PAA, its biosynthesis, inactivation and physiological significance in plants are largely unknown. Here, we demonstrate that overexpression of the CYP79A2 gene, which is involved in benzylglucosinolate synthesis, remarkably increased the levels of PAA and enhanced lateral root formation in Arabidopsis. This coincided with a significant reduction in the levels of IAA. The results from auxin metabolite quantification suggest that the PAA-dependent induction of GH3 genes, which encode auxin-amido synthetases, promote the inactivation of IAA. Similarly, an increase of IAA synthesis, via the indole-acetaldoxime pathway, significantly reduced the levels of PAA. The same adjustment of IAA and PAA levels was also observed by applying each auxin to wild-type plants. These results show that GH3 auxin-amido synthetases can alter the ratio of IAA and PAA in plant growth and development.

Plants take up water and nutrients through roots, and uptake efficiency depends on root behavior. Roots recognize the moisture gradient in soil and grow toward the direction of high moisture. This phenomenon is called hydrotropism and it contributes to efficient water uptake. As nutrients in soil are also unevenly distributed, it is beneficial for plants to grow their roots in the direction of increasing nutrient concentrations, but such a phenomenon has not been demonstrated. Here, we describe the directional growth of roots in response to a nutrient gradient. Using our assay system, gradient of a nitrogen nutrient, NH4+, was sufficient to stimulate positive tropic responses of rice lateral roots. This phenomenon is a tropism of plant roots to nutrients; hence, we propose the name “nutritropism”. As well as other tropisms, differential cell elongation was observed before the elongation zone during nutritropism, but the pattern promoting cell elongation preferentially on the non-stimulated side was opposite to those in root hydrotropism and gravitropism. Our evaluation of the NH4+ gradient suggested that the root tips responded to a sub-micromolar difference in NH4+ concentration on the both sides of the root. Hydrotropism, gravitropism, and phototropism were described in plants as the “power of movement” by Charles and Francis Darwin in 1880, and these three tropisms have attracted the attention of plant scientists for more than 130 years. Our discovery of nutritropism represents the fourth “power of movement” in plants and provides a novel root behavioral property used by plants to acquire nutrients efficiently.

Plant cell walls are sophisticated carbohydrate-rich structures representing the immediate contact surface with the extracellular environment, often serving as the first barrier against biotic and abiotic stresses. Notably, a variety of perturbations in plant cell walls result in upregulated jasmonate (JA) production, a phytohormone with essential roles in defense and growth responses. Hence, cell wall-derived signals can initiate intracellular JA-mediated responses and the elucidation of the underlying signaling pathways could provide novel insights into cell wall maintenance and remodeling, as well as advance our understanding on how is JA biosynthesis initiated. This Mini Review will describe current knowledge about cell wall-derived damage signals and their effects on JA biosynthesis, as well as provide future perspectives.

The phytohormone auxin governs various developmental processes in plants including vascular formation. Auxin transport and biosynthesis are important factors in determining auxin distribution in tissues. Although the role of auxin transport in vein pattern formation is widely recognized, that of auxin biosynthesis in vascular development is poorly understood. Heterodimer complexes comprising two basic helix–loop–helix protein families, LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS5 (TMO5)/TMO5-LIKE1 (T5L1), are master transcriptional regulators of the initial process of vascular development. The LHW–TMO5/T5L1 dimers regulate vascular initial cell production, vascular cell proliferation and xylem fate determination in the embryo and root apical meristem (RAM). In this study, we investigated the function of local auxin biosynthesis in initial vascular development in RAM. Results showed that LHW–T5L1 upregulated the expression of YUCCA4 (YUC4), a key auxin biosynthesis gene. The expression of YUC4 was essential for promoting xylem differentiation and vascular cell proliferation in RAM. Conversely, auxin biosynthesis was required for maintaining the expression levels of LHW, TMO5/T5L1 and their targets. Our results suggest that local auxin biosynthesis forms a positive feedback loop for fine-tuning the level of LHW–TMO5/T5L1, which is necessary for initiating vascular development.

In plants, chlorophyll (Chl) a and b are interconvertible by the action of three enzymes—chlorophyllide a oxygenase, Chl b reductase (CBR) and 7-hydroxymethyl chlorophyll a reductase (HCAR). These reactions are collectively referred to as the Chl cycle. In plants, this cyclic pathway ubiquitously exists and plays essential roles in acclimation to different light conditions at various developmental stages. By contrast, only a limited number of cyanobacteria species produce Chl b, and these include Prochlorococcus, Prochloron, Prochlorothrix and Acaryochloris. In this study, we investigated a possible existence of the Chl cycle in Chl b synthesizing cyanobacteria by testing in vitro enzymatic activities of CBR and HCAR homologs from Prochlorothrix hollandica and Acaryochloris RCC1774. All of these proteins show respective CBR and HCAR activity in vitro, indicating that both cyanobacteria possess the potential to complete the Chl cycle. It is also found that CBR and HCAR orthologs are distributed only in the Chl b-containing cyanobacteria that habitat shallow seas or freshwater, where light conditions change dynamically, whereas they are not found in Prochlorococcus species that usually habitat environments with fixed lighting. Taken together, our results implicate a possibility that the Chl cycle functions for light acclimation in Chl b-containing cyanobacteria.

Auxin is a phytohormone that plays an important role in plant growth and development by forming local concentration gradients. The regulation of auxin levels is determined by the activity of auxin efflux carrier protein PIN-formed (PIN). In Arabidopsis thaliana, PIN-formed1 (PIN1) functions in inflorescence and root development. In rice (Oryza sativa L.), there are four PIN1 homologs (OsPIN1a–1d), but their functions remain largely unexplored. Hence, in this study, we created mutant alleles of PIN1 gene—pin1a, pin1b, pin1c, pin1d, pin1a pin1b and pin1c pin1d— using CRISPR/Cas9 technology and used them to study the functions of the four OsPIN1 paralogs in rice. In wild-type rice, all four OsPIN1 genes were relatively highly expressed in the root than in other tissues. Compared with the wild type, the OsPIN1 single mutants had no dramatic phenotypes, but the pin1a pin1b double mutant had shorter shoots and primary roots, fewer crown roots, reduced root gravitropism, longer root hairs and larger panicle branch angle. Furthermore, the pin1c pin1d double mutant showed no observable phenotype at the seedling stage, but showed naked, pin-shape inflorescence at flowering. These data suggest that OsPIN1a and OsPIN1b are involved in root, shoot and inflorescence development in rice, whereas OsPIN1c and OsPIN1d mainly function in panicle formation. Our study provides basic knowledge that will facilitate the study of auxin transport and signaling in rice.

Abiotic environmental stresses have a negative impact on the yield and quality of crops. Understanding these stresses is an essential enabler for mitigating breeding strategies and it becomes more important as the frequency of extreme weather conditions increases due to climate change. This study analyses the response of barley (Hordeum vulgare L.) to a heat wave during grain filling in three distinct stages: the heat wave itself, the return to a normal temperature regime, and the process of maturation and desiccation. The properties and structure of the starch produced were followed throughout the maturational stages. Furthermore, the key enzymes involved in the carbohydrate supply to the grain were monitored. We observed differences in starch structure with well-separated effects because of heat stress and during senescence. Heat stress produced marked effects on sucrolytic enzymes in source and sink tissues. Early cessation of plant development as an indirect consequence of the heat wave was identified as the major contributor to final yield loss from the stress, highlighting the importance for functional stay-green traits for the development of heat-resistant cereals.

Boea hygrometrica can survive extreme drought conditions and has been used as a model to study desiccation tolerance. A genome-wide transcriptome analysis of B. hygrometrica showed that the plant can survive rapid air-drying after experiencing a slow soil-drying acclimation phase. In addition, a weighted gene co-expression network analysis was used to study the transcriptomic datasets. A network comprising 22 modules was constructed, and seven modules were found to be significantly related to desiccation response using an enrichment analysis. Protein ubiquitination was observed to be a common process linked to hub genes in all the seven modules. Ubiquitin-modified proteins with diversified functions were identified using immunoprecipitation coupled with mass spectrometry. The lowest level of ubiquitination was noted at the full soil drying priming stage, which coincided the accumulation of dehydration-responsive gene BhLEA2. The highly conserved RY motif (CATGCA) was identified from the promoters of ubiquitin-related genes that were downregulated in the desiccated samples. An in silico gene expression analysis showed that the negative regulation of ubiquitin-related genes is potentially mediated via a B3 domain-containing transcription repressor VAL1. This study suggests that priming may involve the transcriptional regulation of several major processes, and the transcriptional regulation of genes in protein ubiquitination may play a hub role to deliver acclimation signals to posttranslational level in the acquisition of desiccation tolerance in B. hygrometrica.

Wasabi (Eutrema japonicum) is a perennial plant native to Japan that is used as a spice because it contains isothiocyanates. It also contains an isosaponarin, 4′-O-glucosyl-6-C-glucosyl apigenin, in its leaves, which has received increasing attention in recent years for its bioactivity, such as its promotion of type-I collagen production. However, its biosynthetic enzymes have not been clarified. In this study, we partially purified a C-glucosyltransferase (CGT) involved in isosaponarin biosynthesis from wasabi leaves and identified the gene coding for it (WjGT1). The encoded protein was similar to UGT84 enzymes and was named UGT84A57. The recombinant enzyme of WjGT1 expressed in Escherichia coli showed C-glucosylation activity toward the 6-position of flavones such as apigenin and luteolin. The enzyme also showed significant activity toward flavonols, but trace or no activity toward flavone 4′-O-glucosides, suggesting that isosaponarin biosynthesis in wasabi plants would proceed by 6-C-glucosylation of apigenin, followed by its 4′-O-glucosylation. Interestingly, the enzyme showed no activity against sinapic acid or p-coumaric acid, which are usually the main substrates of UGT84 enzymes. The accumulation of WjGT1 transcripts was observed mainly in the leaves and flowers of wasabi, in which C-glucosylflavones were accumulated. Molecular phylogenetic analysis suggested that WjGT1 acquired C-glycosylation activity independently from other reported CGTs after the differentiation of the family Brassicaceae.

SCO (synthesis of cytochrome c oxidase) proteins are involved in the insertion of copper during the assembly of cytochrome c oxidase (COX), the final enzyme of the mitochondrial respiratory chain. Two SCO proteins, namely, homolog of copper chaperone 1 and 2 (HCC1 and HCC2) are present in seed plants, but HCC2 lacks the residues involved in copper binding, leading to uncertainties about its function. In this study, we performed a transcriptomic and phenotypic analysis of Arabidopsis thaliana plants with reduced expression of HCC1 or HCC2. We observed that a deficiency in HCC1 causes a decrease in the expression of several stress-responsive genes, both under basal growth conditions and after applying a short-term high salinity treatment. In addition, HCC1 deficient plants show a faster decrease in chlorophyll content, photosystem II quantum efficiency and COX levels after salinity stress, as well as a faster increase in alternative oxidase capacity. Notably, HCC2 deficiency causes opposite changes in most of these parameters. Bimolecular fluorescence complementation analysis indicated that both proteins are able to interact. We postulate that HCC1 is a limiting factor for COX assembly during high salinity conditions and that HCC2 probably acts as a negative modulator of HCC1 activity through protein–protein interactions. In addition, a direct or indirect role of HCC1 and HCC2 in the gene expression response to stress is proposed.

Microalgal ice-binding proteins (IBPs) in the polar region are poorly understood at the genome-wide level, although they are important for cold adaptation. Through the transcriptome study with the Arctic green alga Chloromonas sp. KNF0032, we identified six Chloromonas IBP genes (CmIBPs), homologous with the previously reported IBPs from Antarctic snow alga CCMP681 and Antarctic Chloromonas sp. They were organized with multiple exon/intron structures and low-temperature-responsive cis-elements in their promoters and abundantly expressed at low temperature. The biological functions of three representative CmIBPs (CmIBP1, CmIBP2 and CmIBP3) were tested using in vitro analysis and transgenic plant system. CmIBP1 had the most effective ice recrystallization inhibition (IRI) activities in both in vitro and transgenic plants, and CmIBP2 and CmIBP3 had followed. All transgenic plants grown under nonacclimated condition were freezing tolerant, and especially 35S::CmIBP1 plants were most effective. After cold acclimation, only 35S::CmIBP2 plants showed slightly increased freezing tolerance. Structurally, the CmIBPs were predicted to have β-solenoid forms with parallel β-sheets and repeated TXT motifs. The repeated TXT structure of CmIBPs appears similar to the AidA domain-containing adhesin-like proteins from methanogens. We have shown that the AidA domain has IRI activity as CmIBPs and phylogenetic analysis also supported that the AidA domains are monophyletic with ice-binding domain of CmIBPs, and these results suggest that CmIBPs are a type of modified adhesins.

As sessile and autotrophic organisms, plants have evolved sophisticated pathways to produce a rich array of specialized metabolites, many of which are biologically active and function as defense substances in protecting plants from herbivores and pathogens. Upon stimuli, these structurally diverse small molecules may be synthesized or constitutively accumulated. Jasmonate acids (JAs) are the major defense phytohormone involved in transducing external signals (such as wounding) to activate defense reactions, including, in particular, the reprogramming of metabolic pathways that initiate and enhance the production of defense compounds against insect herbivores and pathogens. In this review, we summarize the progress of recent research on the control of specialized metabolic pathways in plants by JA signaling, with an emphasis on the molecular regulation of terpene and alkaloid biosynthesis. We also discuss the interplay between JA signaling and various signaling pathways during plant defense responses. These studies provide valuable data for breeding insect-proof crops and pave the way to engineering the production of valuable metabolites in future.

Phosphorus is one of the most important macronutrients required for plant growth and development. The importance of phosphorylation modification in regulating phosphate (Pi) homeostasis in plants is emerging. We performed phosphoproteomic profiling to characterize proteins whose degree of phosphorylation is altered in response to Pi starvation in rice root. A subset of 554 proteins, including 546 down-phosphorylated and eight up-phosphorylated proteins, exhibited differential phosphorylation in response to Pi starvation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the differentially phosphorylated proteins indicated that RNA processing, transport, splicing and translation and carbon metabolism played critical roles in response to Pi starvation in rice. Levels of phosphorylation of four mitogen-activated protein kinases (MAPKs), including OsMAPK6, five calcium-dependent protein kinases (CDPKs) and OsCK2β3 decreased in response to Pi starvation. The decreased phosphorylation level of OsMAPK6 was confirmed by Western blotting. Mutation of OsMAPK6 led to Pi accumulation under Pi-sufficient conditions. Motif analysis indicated that the putative MAPK, casein kinase 2 (CK2) and CDPK substrates represented about 54.4%, 21.5% and 4.7%, respectively, of the proteins exhibiting differential phosphorylation. Based on the motif analysis, 191, 151 and 46 candidate substrates for MAPK, CK2 and CDPK were identified. These results indicate that modification of phosphorylation profiles provides complementary information on Pi-starvation-induced processes, with CK2, MAPK and CDPK protein kinase families playing key roles in these processes in rice.

Abscisic acid (ABA) is a phytohormone and a major determinant of seed dormancy in plants. Seed dormancy is gradually lost during dry storage, a process known as ‘after-ripening’, and this dormancy decay is related to a decline in ABA content and sensitivity in seeds after imbibition. In this study, we aimed at investigating the effect of after-ripening on ABA signaling in barley, our cereal model species. Phosphosignaling networks in barley grains were investigated by a large-scale analysis of phosphopeptides to examine potential changes in response pathways to after-ripening. We used freshly harvested (FH) and after-ripened (AR) barley grains which showed different ABA sensitivity. A total of 1,730 phosphopeptides were identified in barley embryos isolated from half-cut grains. A comparative analysis showed that 329 and 235 phosphopeptides were upregulated or downregulated, respectively after ABA treatment, and phosphopeptides profiles were quite different between FH and AR embryos. These results were supported by peptide motif analysis which suggested that different sets of protein kinases are active in FH and AR grains. Furthermore, in vitro phosphorylation assays confirmed that some phosphopeptides were phosphorylated by SnRK2s, which are major protein kinases involved in ABA signaling. Taken together, our results revealed very distinctive phosphosignaling networks in FH and AR embryos of barley, and suggested that the after-ripening of barley grains is associated with differential regulation of phosphosignaling pathways leading to a decay of ABA signaling.

The biosynthesis and roles of strigolactones (SLs) have been investigated in herbaceous plants, but so far, their role in trees has received little attention. In this study, we analyzed the presence, spatial/temporal expression and role of SL pathway genes in Populus tremula × Populus tremuloides. In this proleptic species, axillary buds (AXBs) become para-dormant at the bud maturation point, providing an unambiguous starting point to study AXB activation. We identified previously undescribed Populus homologs of DWARF27 (D27), LATERAL BRANCHING OXIDOREDUCTASE (LBO) and DWARF53-like (D53-like) and analyzed the relative expression of all SL pathway genes in root tips and shoot tissues. We found that, although AXBs expressed MORE AXILLARY GROWTH1 (MAX1) and LBO, they did not express MAX3 and MAX4, whereas nodal bark expressed high levels of all SL biosynthesis genes. By contrast, expression of the SL perception and signaling genes MAX2, D14 and D53 was high in AXBs relative to nodal bark and roots. This suggests that AXBs are reliant on the associated nodes for the import of SLs and SL precursors. Activation of AXBs was initiated by decapitation and single-node isolation. This rapidly downregulated SL pathway genes downstream of MAX4, although later these genes were upregulated coincidently with primordia formation. GR24-feeding counteracted all activation-related changes in SL gene expression but did not prevent AXB outgrowth showing that SL is ineffective once AXBs are activated. The results indicate that nodes rather than roots supply SLs and its precursors to AXBs, and that SLs may restrain embryonic shoot elongation during AXB formation and para-dormancy in intact plants.

In Artemisia annua plants, glandular trichomes (GTs) are responsible for the biosynthesis and secretion of sesquiterpene lactones including artemisinin/arteannuin B. Nonspecific lipid transfer proteins (LTPs) in plants bind and carry lipid molecules across the cell membrane and are also known as secretary proteins. Interestingly, the transcripts of LTP genes are exceptionally abundant in the GTs of A. annua. In the present study, we isolated two trichome-specific LTP genes (AaLTP3 and AaLTP4) from a Korean ecotype of A. annua. AaLTP3 was expressed abundantly in shoots, whereas AaLTP4 was expressed in flowers. The GUS signal driven by the AaLTP3 or AaLTP4 promoter in transgenic A. annua plants revealed that the AaLTP3 promoter was active on hair-like non-GTs and that the AaLTP4 promoter was active on GTs. Analysis of enhanced cyan fluorescent protein (ECFP) fluorescence fused with the AaLTP3 or AaLTP4 protein in transgenic tobacco revealed that ECFP florescence was very bright on secreted lipids of long GTs. Moreover, the florescence was also bright on the head cells of short trichomes and their secreted granules. Immunoblotting analysis of GT exudates in petioles of A. annua revealed a strong positive signal against the AaLTP4 antibody. Overexpression of AaLTP3 or AaLTP4 in transgenic A. annua plants resulted in enhanced production of sesquiterpene lactones (arteannuin B, artemisinin, dihydroartemisinic acid and artemisinic acid) compared with those of wild type. The present study shows that LTP genes (AaLTP3 or AaLTP4) play important roles in the sequestration and secretion of lipids in GTs of A. annua, which is useful for the enhanced production of sesquiterpene lactones by genetic engineering.

Production of vegetable oils is a vital agricultural resource and oilseed rape (Brassica napus) is the third most important oil crop globally. Although the regulation of lipid biosynthesis in oilseeds is still not fully defined, the acyl-CoA-binding proteins (ACBPs) have been reported to be involved in such metabolism, including oil accumulation, in several plant species. In this study, progressive changes in gene expression in embryos and seed coats at different stages of seed development were comprehensively investigated by transcriptomic analyses in B. napus, revealing dynamic changes in the expression of genes involved in lipid biosynthesis. We show that genes encoding BnACBP proteins show distinct changes in expression at different developmental stages of seed development and show markedly different expression between embryos and seed coats. Both isoforms of the ankyrin-repeat BnACBP2 increased during the oil accumulation period of embryo development. By contrast, the expression of the three most abundant isoforms of the small molecular mass BnACBP6 in embryos showed progressive reduction, despite having the highest overall expression level. In seed coats, BnACBP3, BnACBP4 and BnACBP5 expression remained constant during development, whereas the two major isoforms of BnACBP6 increased, contrasting with the data from embryos. We conclude that genes related to fatty acid and triacylglycerol biosynthesis showing dynamic expression changes may regulate the lipid distribution in embryos and seed coats of B. napus and that BnACBP2 and BnACBP6 are potentially important for oil accumulation.

Regulation of defense and developmental responses by jasmonates (JAs) has been intensively investigated at genetic and transcriptional levels. Plasticity in the jasmonic acid (JA) metabolic pathway as a means to control signal output has received less attention. Although the amplitude of JA responses generally follows the accumulation dynamics of the active hormone jasmonoyl-isoleucine (JA-Ile), emerging evidence has identified cases where this relationship is distorted and that we discuss in this review. JA-Ile is turned over in Arabidopsis by two inducible, intertwined catabolic pathways; one is oxidative and mediated by cytochrome P450 enzymes of the subfamily 94 (CYP94), and the other proceeds via deconjugation by amidohydrolases. Their genetic inactivation has profound effects on JAs homeostasis, including strong JA-Ile overaccumulation, but this correlates with enhanced defense and tolerance to microbial or insect attacks only in the absence of overinduction of negative signaling regulators. By contrast, the impairment of JA oxidation in the jasmonic acid oxidase 2 (jao2) mutant turns on constitutive defense responses without elevating JA-Ile levels in naive leaves and enhances resistance to subsequent biotic stress. This latter and other recent cases of JA signaling are associated with JA-Ile catabolites accumulation rather than more abundant hormone, reflecting increased metabolic flux through the pathway. Therefore, manipulating upstream and downstream JA-Ile homeostatic steps reveals distinct metabolic nodes controlling defense signaling output.

The lipid-derived hormones jasmonates (JAs) play key functions in a wide range of physiological and developmental processes that regulate growth, secondary metabolism and defense against biotic and abiotic stresses. In this connection, biosynthesis, tissue-specific distribution, metabolism, perception, signaling of JAs have been the target of extensive studies. In recent years, the involvement of JAs signaling pathway in the regulation of growth and adaptive responses to environmental challenges has been further examined. However, JAs-mediated mechanisms underlying the transition from ‘growth mode’ to ‘adaptive mode’ remain ambiguous. Combined analysis of transgenic lines deficient in JAs signaling in conjunction with the data from JAs-treated plants revealed the function of these hormones in rewiring of central metabolism. The collective data illustrate JAs-mediated decrease in the levels of metabolites associated with active growth such as sucrose, raffinose, orotate, citrate, malate, and an increase in phosphorylated hexoses, responsible for the suppression of growth and photosynthesis, concurrent with the induction of protective metabolites, such as aromatic and branched-chain amino acids, and aspartate family of metabolites. This finding provides an insight into the function of JAs in shifting the central metabolism from the production of growth-promoting metabolites to protective compounds and expands our understanding of the role of JAs in resource allocation in response to environmental challenges.

DefenseDevelopmentJasmonateMetabolismReproductionSignaling

Plants convert solar energy into chemical energy through photosynthesis, which supports almost all life activities on earth. Because the intensity and quality of sunlight can change dramatically throughout the day, various regulatory mechanisms help plants adjust their photosynthetic output accordingly, including the regulation of light energy accumulation to prevent the generation of damaging reactive oxygen species. Non-photochemical quenching (NPQ) is a regulatory mechanism that dissipates excess light energy, but how it is regulated is not fully elucidated. In this study, we report a new NPQ-regulatory protein named Day-Length-dependent Delayed-Greening1 (DLDG1). The Arabidopsis DLDG1 associates with the chloroplast envelope membrane, and the dldg1 mutant had a large NPQ value compared with wild type. The mutant also had a pale-green phenotype in developing leaves but only under continuous light; this phenotype was not observed when dldg1 was cultured in the dark for ≥8 h/d. DLDG1 is a homolog of the plasma membrane-localizing cyanobacterial proton-extrusion-protein A that is required for light-induced H+ extrusion and also shows similarity in its amino-acid sequence to that of Ycf10 encoded in the plastid genome. Arabidopsis DLDG1 enhances the growth-retardation phenotype of the Escherichia coli K+/H+ antiporter mutant, and the everted membrane vesicles of the E. coli expressing DLDG1 show the K+/H+ antiport activity. Our findings suggest that DLDG1 functionally interacts with Ycf10 to control H+ homeostasis in chloroplasts, which is important for the light-acclimation response, by optimizing the extent of NPQ.

The last stages of stamen development, collectively called stamen maturation, encompass pollen viability, filament elongation and anther dehiscence or opening. These processes are essential for male fertility in Arabidopsis and require the function of jasmonate signaling. There is a good understanding of jasmonate synthesis, perception and transcriptional outputs in Arabidopsis stamens. In addition, the spatiotemporal localization of jasmonate signaling components at the tissue and cellular levels has started to emerge in recent years. However, the ultimate cellular functions activated by jasmonate to promote stamen maturation remain unknown. The hormones auxin and gibberellin have been proposed to control the activation of jasmonate synthesis to promote stamen maturation, although we hypothesize that this action is rather indirect. In this review, we examine these different areas, attempt to clarify some confusing aspects found in the literature and raise testable hypothesis that may help to further understand how jasmonate controls male fertility in Arabidopsis.

Light is one of the most essential environmental clues for plant growth and morphogenesis. Exposure to blue monochromatic light from darkness is a turning point for plant biological activity and as a result dramatic changes in gene expression occur. To understand the translational impacts of blue light, we have performed ribosome profiling analysis and called translated ORFs de novo within not only mRNAs but also non-coding RNAs. Translation efficiency of 3,823 protein-coding ORFs, such as nuclear chloroplast-related genes, was up-regulated by blue light exposure. Moreover, the translational activation of the microRNA biogenesis-related genes, DCL1 and HYL1, was induced by blue light. Considering the 3-nt codon periodicity of ribosome footprints, a few hundred short ORFs (sORFs) lying on non-coding RNAs (ncRNAs) and upstream ORFs (uORFs) on mRNAs were found that had differential translation status between blue light and dark. uORFs are known to have a negative effect on expression of the main ORFs (mORFs) on the same mRNAs. Our analysis suggests that translation of uORFs is likely to be more stimulated than that of the corresponding mORFs, and uORF-mediated translational repression of the mORFs in five genes was induced by blue light exposure. With data-based annotation of the ORFs, our analysis provides insights into the translatome in response to environmental changes, such as those involving light.