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SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes encode a large family of plant-specific transcription factors that play important roles in plant growth, development, and stress responses. However, there is little information available on SPL genes in Chenopodiaceae. Here, 23 SPL genes were identified and characterized in the highly nutritious crop Chenopodium quinoa. Chromosome localization analysis indicated that the 23 CqSPL genes were unevenly distributed on 12 of 18 chromosomes. Two zinc finger-like structures and a nuclear location signal were present in the SBP domains of all CqSPLs, with the exception of CqSPL21/22. Phylogenetic analysis revealed that these genes were classified into eight groups (group I–VIII). The exon–intron structure and motif composition of the genes in each group were similar. Of the 23 CqSPLs, 13 were potential targets of miR156/7. In addition, 5 putative miR156-encoding loci and 13 putative miR157-encoding loci were predicted in the quinoa genome, and they were unevenly distributed on chromosome 1–4. The expression of several Cqu-MIR156/7 loci was confirmed by reverse transcription polymerase chain reaction in seedlings. Many putative cis-elements associated with light, stress, and phytohormone responses were identified in the promoter regions of CqSPLs, suggesting that CqSPL genes are likely involved in the regulation of key developmental processes and stress responses. Expression analysis revealed highly diverse expression patterns of CqSPLs among tissues. Many CqSPLs were highly expressed in leaves, flowers, and seeds, and their expression levels were low in the roots, suggesting that CqSPLs play distinct roles in the development and growth of quinoa. The expression of 13 of 23 CqSPL genes responded to salt treatment (11 up-regulated and 2 down-regulated). A total of 22 of 23 CqSPL genes responded to drought stress (21 up-regulated and 1 down-regulated). Moreover, the expression of 14 CqSPL genes was significantly altered following cadmium treatment (3 up-regulated and 11 down-regulated). CqSPL genes are thus involved in quinoa responses to salt/drought and cadmium stresses. These findings provide new insights that will aid future studies of the biological functions of CqSPLs in C. quinoa.

BACKGROUND: PPO (prophenoloxidase) cascade plays an important role in resisting invasion of entomogenous fungus. The 20‐hydroxyecdysone (20E) exerts potent effect on the innate immunity in many insects. However, whether 20E controls the PPO cascade system against fungi and the regulatory mechanism in insects remains unclear. RESULTS: In this study, both the proteome and transcriptome of Locusta migratoria were determined followed by the induction of 20E. Pattern recognition receptor GNBP‐2 (Gram‐negative binding proteins) has been identified that responded to 20E at both messenger RNA (mRNA) and protein levels. The PPO gene expression in fat body and PO (phenoloxidase) activity in plasma was found significantly induced after 20E injection and during the high‐20E developmental stage. However, when 20E signal was blocked by RNA interference (RNAi) of ecdysone receptor, the expression level of PPO and PO activity failed to be increased by 20E. Thus, 20E could not significantly induce the expression of PPO gene and PO activity after RNAi of GNBP‐2. Furthermore, 20E treatment notably enhanced the resistance of L. migratoria against Metarhizium anisopliae. Followed by of GNBP‐2 silencing, the mortality of nymphs was significantly increased under the stress of Metarhizium anisopliae, and 20E injection could not increase the resistance. CONCLUSION: The 20E regulates the PPO system to resist fungal invasion via regulating GNBP‐2 in worldwide pest L. migratoria. Our results provide insight into the mechanism of how 20E enhances the antimicrobial immunity, and will be beneficial for modification of entomogenous fungi targeting on hormones and the immune system. © 2020 Society of Chemical Industry

Nickel (Ni) contamination imposes deleterious effects on the stability of soil ecosystem. Soil fungal community as a crucial moderator of soil remediation and biochemical processes has attracted more and more research interests. In the present study, soil fungal community composition and diversity under long-term Ni contamination were investigated and fungal interaction networks were built to reveal fungal co-occurrence patterns. The results showed that moderate Ni contamination significantly increased fungal diversity and altered fungal community structure. Functional predictions based on FUNGuild suggested that the relative abundance of arbuscular mycorrhizal fungi (AMF) significantly increased at moderate Ni contamination level. Ni contamination strengthened fungal interactions. Keystone taxa at different Ni contamination levels, such as Penicillium at light contamination, were identified, which might have ecological significance in maintaining the stability of fungal community to Ni stress. The present study provided a deeper insight into the effect of long-term Ni contamination on fungal community composition and co-occurrence patterns, and was helpful to further explore ecological risk of Ni contamination in cultivated field.

Significance The GET pathway is required for the insertion of tail-anchored (TA) membrane proteins in the endoplasmic reticulum (ER) of yeast and mammals. Some orthologous genes had also been identified in higher plants with the exception of one of the two ER membrane receptors required for membrane insertion. Get2/CAML is required for the pathway’s cytosolic chaperone to dock and release its TA protein cargo. Here we report the identification of the elusive plant GET pathway receptor through an interaction screen in Arabidopsis. The candidate allows detection of further Get2/CAML orthologs in higher plants, revealing conservation and function of structural features across kingdoms. Additionally, our results demonstrate that these features, rather than sequence conservation, determine functionality of the candidate within the pathway., Type II tail-anchored (TA) membrane proteins are involved in diverse cellular processes, including protein translocation, vesicle trafficking, and apoptosis. They are characterized by a single C-terminal transmembrane domain that mediates posttranslational targeting and insertion into the endoplasmic reticulum (ER) via the Guided-Entry of TA proteins (GET) pathway. The GET system was originally described in mammals and yeast but was recently shown to be partially conserved in other eukaryotes, such as higher plants. A newly synthesized TA protein is shielded from the cytosol by a pretargeting complex and an ATPase that delivers the protein to the ER, where membrane receptors (Get1/WRB and Get2/CAML) facilitate insertion. In the model plant Arabidopsis thaliana, most components of the pathway were identified through in silico sequence comparison, however, a functional homolog of the coreceptor Get2/CAML remained elusive. We performed immunoprecipitation-mass spectrometry analysis to detect in vivo interactors of AtGET1 and identified a membrane protein of unknown function with low sequence homology but high structural homology to both yeast Get2 and mammalian CAML. The protein localizes to the ER membrane, coexpresses with AtGET1, and binds to Arabidopsis GET pathway components. While loss-of-function lines phenocopy the stunted root hair phenotype of other Atget lines, its heterologous expression together with the coreceptor AtGET1 rescues growth defects of Δget1get2 yeast. Ectopic expression of the cytosolic, positively charged N terminus is sufficient to block TA protein insertion in vitro. Our results collectively confirm that we have identified a plant-specific GET2 in Arabidopsis, and its sequence allows the analysis of cross-kingdom pathway conservation.

Simple Summary Insect reproduction is an important and complicated process required for producing healthy individuals and maintaining their population abundance. Thus, it could become a valuable target for insect biological control. To date, many factors and pathways have been revealed to be involved in this reproductive process, but it is still far from a full understanding of the molecular network underlying this process. We herein investigated a RNA helicase, DEAD-box protein 6 (DDX6) in Locusta migratoria, a global, destructive pest, and found that knockdown LmDDX6 downregulated expression levels of juvenile hormone receptor gene methoprene-tolerant and its target genes 78-kDa glucose-regulated proteins, thus reducing vitellogenin expression and ultimately impairing the ovary development and oocyte maturation. These results demonstrate that LmDDX6 is a key player in female locust reproduction, providing, thus, a novel target for locust biological control. Abstract DEAD-box protein 6 (DDX6) is a member of the DDX RNA helicase family that exists in all eukaryotes. It has been extensively studied in yeast and mammals and has been shown to be involved in messenger ribonucleoprotein assembly, mRNA storage, and decay, as well as in miRNA-mediated gene silencing. DDX6 participates in many developmental processes but the biological function of DDX6 in insects has not yet been adequately addressed. Herein, we characterized the LmDDX6 gene that encodes the LmDDX6 protein in Locusta migratoria, a global, destructive pest. LmDDX6 possesses five motifs unique to the DDX6 subfamily. In the phylogenetic tree, LmDDX6 was closely related to its orthologs in Apis dorsata and Zootermopsis nevadensis. RT-qPCR data revealed high expression of LmDDX6 in the ovary, muscle, and fat body, with a declining trend in the ovary after adult ecdysis. LmDDX6 knockdown downregulated the expression levels of the juvenile hormone receptor Met, and genes encoding Met downstream targeted Grp78-1 and Grp78-2, reduced LmVg expression, and impaired ovary development and oocyte maturation. These results demonstrate that LmDDX6 plays an essential role in locust female reproduction and, thus, could be a novel target for locust biological control.

Chromium (Cr) pollution has attracted much attention due to its biological toxicity. However, little is known regarding Cr toxicity to soil microorganisms. The present study assesses the toxicity of Cr(VI) on two microbial processes, potential nitrification rate (PNR) and substrate-induced respiration (SIR), in a wide range of agricultural soils and detected the abundance of soil bacteria, fungi, ammonia-oxidizing bacteria and archaea. The toxicity thresholds of 10% and 50% effective concentrations (EC10 and EC50) for PNR varied by 32.18- and 38.66-fold among different soils, while for SIR they varied by 391.21- and 16.31-fold, respectively. Regression model analysis indicated that for PNR, CEC as a single factor explained 27% of the variation in EC10, with soil clay being the key factor explaining 47.3% of the variation in EC50. For SIR, organic matter and pH were found to be the most vital predictors for EC10 and EC50, explaining 34% and 61.1% of variation, respectively. In addition, extended aging time was found to significantly attenuate the toxicity of Cr on PNR. SIR was mainly driven by total bacteria rather than fungi, while PNR was driven by both AOA and AOB. These results were helpful in deriving soil Cr toxicity threshold based on microbial processes, and provided a theoretical foundation for ecological risk assessments and establishing a soil environmental quality criteria for Cr.

DDX3 represents a well-defined subfamily of DEAD-box RNA helicase and exerts multiple functions in RNA metabolism, cell cycle, tumorigenesis, signal pathway, and fertility. Our previous study has shown that LmDDX3, the ortholog of DDX3 in Locusta migratoria, is ubiquitously expressed, and with a high abundance in testis and ovary. Knockdown of LmDDX3 results in a lethal phenotype in nymph, but it still remains unclear for its role in reproductive process. In this study, we therefore characterized LmDDX3 expression in female adult locust and analyzed its function in oocyte development. LmDDX3 was expressed in all tissues examined with significant more transcripts in ovary and hindgut. In ovary, a strong expression level was detected at the day just after adult eclosion, and a dramatic reduction then occurred during the oocyte development. LmDDX3 RNAi led to a reduced vitellogenin (Vg) expression in fat body via partially at least, the JH signaling pathway, and caused an upregulation of vitellogenin receptor (VgR) in ovary, and thus blocked the ovarian development and oocyte maturation. Sequence and phylogenetic analysis indicated that LmDDX3 was closely related to termite DDX3. Taken together, these data reveal a critical role for LmDDX3 in regulating the transcription of Vg and VgR, two major factors in vitellogenesis that is a key process required for ovary development and oocyte maturation in locust, and contribute thereof a new putative target for locust biological control.

Copper (Cu) contamination threatens the stability of soil ecosystems. As important moderators of biochemical processes and soil remediation, the fungal community in contaminated soils has attracted much research interest. In this study, soil fungal diversity and community composition under long-term Cu contamination were investigated based on high-throughput sequencing. The co-occurrence networks were also constructed to display the co-occurrence patterns of the soil fungal community. The results showed that the richness and Chao1 index both significantly increased at 50 mg kg−1 Cu and then significantly decreased at 1600 and 3200 mg kg−1 Cu. Soil fungal diversity was significantly and positively correlated with plant dry weight. Specific tolerant taxa under different Cu contamination gradients were illustrated by linear discriminant analysis effect size (LEfSe). Soil Cu concentration and shoot dry weight were the strongest driving factors influencing fungal composition. The relative abundance of arbuscular mycorrhizal fungi increased first and then declined along with elevating Cu concentrations via FUNGuild analysis. The interactions among fungi were enhanced under light and moderate Cu contamination but weakened under heavy Cu contamination by random matrix theory (RMT)-based molecular ecological network analysis. Penicillium, identified as a keystone taxon in Cu-contaminated soils, had the function of removing heavy metals and detoxification, which might be vital to trigger the resistance of the fungal community to Cu contamination. The results may facilitate the identification of Cu pollution indicators and the development of in situ bioremediation technology for contaminated cultivated fields.

Simple Summary Locusta migratoria is a serious agricultural pest all over the world, which devastates agriculture and challenges food security and livelihoods. Recently, RNA interference (RNAi) technology has emerged as a novel strategy for managing insect pests. However, different insect species show a significant variation in RNAi efficiency. Therefore, it is necessary to better understand the RNAi mechanism. In this study, we identified an LmR2D2 gene in the L. migratoria transcriptome. After the expression of LmR2D2 was suppressed by RNAi, we found a significantly diminished RNAi efficiency against a marker gene in L. migratoria. Our binding experiments further demonstrated that the LmR2D2 protein can bind double-stranded RNA (dsRNA) in vitro. On the basis of these results, we conclude that LmR2D2 is involved in the L. migratoria siRNA pathway. Abstract Small interfering RNAs (siRNAs) are non-coding RNAs with a length of 21~23 nucleotides (nt) and present in almost all eukaryotes. The formation of siRNA is a highly conserved post-transcriptional gene-silencing mechanism mediated by key proteins, including Dicer2, Argonaute2 (Ago2) and R2D2. R2D2 has been identified as a double-stranded RNA (dsRNA)-binding protein and reported as an integral component of the siRNA pathway in Drosophila. However, the involvement of R2D2 in the siRNA pathway of Locusta migratoria is still unknown. In the present study, we identified an LmR2D2 gene from the transcriptome of L. migratoria. It consists of a 954-bp open reading frame that encodes a protein of 318 amino acid residues. Further sequence analysis revealed that LmR2D2 possesses two tandem dsRNA-binding domains (dsRBD) at the N-terminus. Analysis of the developmental expression profile of LmR2D2 indicated that its transcript level was stable in third-instar nymphs of L. migratoria, whereas the tissue-dependent expression profile exhibited high levels of expression of LmR2D2 in the testis and ovary. When LmR2D2 was silenced by RNAi, the RNAi efficiency against Lmβ-tubulin as a marker gene was significantly diminished, as indicated by the 37.7% increased Lmβ-tubulin transcript level. Additionally, the prokaryotic expression system was used to obtain the LmR2D2 supernatant protein. By incubating the LmR2D2 protein with biotin-dsRNA, we found that LmR2D2 can bind to dsRNA in vitro, which supports our conclusion that LmR2D2 plays an essential role in the siRNA pathway of L. migratoria.

DEAD-box protein 6 (DDX6) is a member of the DDX RNA helicase family that exists in all eukaryotes. It has been extensively studied in yeast and mammals and has been shown to be involved in messenger ribonucleoprotein assembly, mRNA storage, and decay, as well as in miRNA-mediated gene silencing. DDX6 participates in many developmental processes but the biological function of DDX6 in insects has not yet been adequately addressed. Herein, we characterized the LmDDX6 gene that encodes the LmDDX6 protein in Locusta migratoria, a global, destructive pest. LmDDX6 possesses five motifs unique to the DDX6 subfamily. In the phylogenetic tree, LmDDX6 was closely related to its orthologs in Apis dorsata and Zootermopsis nevadensis. RT-qPCR data revealed high expression of LmDDX6 in the ovary, muscle, and fat body, with a declining trend in the ovary after adult ecdysis. LmDDX6 knockdown downregulated the expression levels of the juvenile hormone receptor Met, and genes encoding Met downstream targeted Grp78-1 and Grp78-2, reduced LmVg expression, and impaired ovary development and oocyte maturation. These results demonstrate that LmDDX6 plays an essential role in locust female reproduction and, thus, could be a novel target for locust biological control.

DEAD-box (DDX) genes encode a group of RNA helicases that are highly conserved and ubiquitously expressed from prokaryotes to eukaryotes, and appear to participate in almost every aspect of RNA metabolism. Studies have been extensively done in yeast and human, in insect, beyond the flies, however, the information of these genes is limited. Here, we therefore identified and characterized 32 DDX genes from Locusta migratoria (L. migratoria), a crop pest. Overview of the gene structure and domain composition showed that the gene size varies significantly from one to fifteen exons, and the encoded proteins contain the conserved helicase core with various extensions at their amino and carboxyl termini. Phylogenetic trees informed that these locust DDX family members have orthologs in all insect species examined and can be classified into 30 subfamilies, all of them found counterparts in human, and most in yeast as well. Quantitative real-time PCR revealed that these genes are expressed in all stages and tissues examined, overall with higher expression level at second and third-instar nymphs and in the reproductive organs. RNA interference (RNAi) analyses showed that seven genes cause lethal phenotype when silenced, of which five lead to defective midgut and gastric caecum, indicating that these genes are essential for the survival and maintenance of normal digestive organs of locust. These data provide a foundation for further functional analysis of these DDX genes in locust.

Next Section Abstract Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins are key players in cellular trafficking and coordinate vital cellular processes, such as cytokinesis, pathogen defense, and ion transport regulation. With few exceptions, SNAREs are tail-anchored (TA) proteins, bearing a C-terminal hydrophobic domain that is essential for their membrane integration. Recently, the Guided Entry of Tail-anchored proteins (GET) pathway was described in mammalian and yeast cells that serve as a blueprint of TA protein insertion [Schuldiner M, et al. (2008) Cell 134(4):634–645; Stefanovic S, Hegde RS (2007) Cell 128(6):1147–1159]. This pathway consists of six proteins, with the cytosolic ATPase GET3 chaperoning the newly synthesized TA protein posttranslationally from the ribosome to the endoplasmic reticulum (ER) membrane. Structural and biochemical insights confirmed the potential of pathway components to facilitate membrane insertion, but the physiological significance in multicellular organisms remains to be resolved. Our phylogenetic analysis of 37 GET3 orthologs from 18 different species revealed the presence of two different GET3 clades. We identified and analyzed GET pathway components in Arabidopsis thaliana and found reduced root hair elongation in Atget lines, possibly as a result of reduced SNARE biogenesis. Overexpression of AtGET3a in a receptor knockout (KO) results in severe growth defects, suggesting presence of alternative insertion pathways while highlighting an intricate involvement for the GET pathway in cellular homeostasis of plants.

Identifying key players and their interactions is fundamental for understanding biochemical mechanisms at the molecular level. The ever-increasing number of alternative ways to detect protein-protein interactions (PPIs) speaks volumes about the creativity of scientists in hunting for the optimal technique. PPIs derived from single experiments or high-throughput screens enable the decoding of binary interactions, the building of large-scale interaction maps of single organisms, and the establishment of cross-species networks. This review provides a historical view of the development of PPI technology over the past three decades, particularly focusing on in vivo PPI techniques that are inexpensive to perform and/or easy to implement in a state-of-the-art molecular biology laboratory. Special emphasis is given to their feasibility and application for plant biology as well as recent improvements or additions to these established techniques. The biology behind each method and its advantages and disadvantages are discussed in detail, as are the design, execution, and evaluation of PPI analysis. We also aim to raise awareness about the technological considerations and the inherent flaws of these methods, which may have an impact on the biological interpretation of PPIs. Ultimately, we hope this review serves as a useful reference when choosing the most suitable PPI technique.

Glutaredoxins (GRXs) are ubiquitous oxidoreductases that play a crucial role in response to oxidative stress by reducing disulfides in various organisms. In planta, three different GRX classes have been identified according to their active site motifs. CPYC and CGFS classes are found in all organisms, whereas the CC-type class is specific for higher land plants. Recently, two Arabidopsis CC-type GRXs, ROXY1 and ROXY2, were shown to exert crucial functions in petal and anther initiation and differentiation. To analyze the function of CC-type GRXs in the distantly related monocots, we isolated and characterized OsROXY1 and OsROXY2—two rice homologs of ROXY1. Both genes are expressed in vegetative and reproductive stages. Although rice flower morphology is distinct from eudicots, OsROXY1/2 floral expression patterns are similar to their Arabidopsis counterparts ROXY1/2. Complementation experiments demonstrate that OsROXY1 and OsROXY2 can fully rescue the roxy1 floral mutant phenotype. Overexpression of OsROXY1, OsROXY2, and ROXY1 in Arabidopsis causes similar vegetative and reproductive plant developmental defects. ROXY1 and its rice homologs thus exert a conserved function during eudicot and monocot flower development. Strikingly, overexpression of these CC-type GRXs also leads to an increased accumulation of hydrogen peroxide levels and hyper-susceptibility to infection from the necrotrophic pathogen Botrytis cinerea, revealing the importance of balanced redox processes in flower organ development and pathogen defence.

We isolated three alleles of an Arabidopsis thaliana gene named ROXY1, which initiates a reduced number of petal primordia and exhibits abnormalities during further petal development. The defects are restricted to the second whorl of the flower and independent of organ identity. ROXY1 belongs to a subgroup of glutaredoxins that are specific for higher plants and we present data on the first characterization of a mutant from this large Arabidopsis gene family for which information is scarce. ROXY1 is predominantly expressed in tissues that give rise to new flower primordia, including petal precursor cells and petal primordia. Occasionally, filamentous organs with stigmatic structures are formed in the second whorl of the roxy1 mutant, indicative for an ectopic function of the class C gene AGAMOUS (AG). The function of ROXY1 in the negative regulation of AG is corroborated by premature and ectopic AG expression in roxy1-3 ap1-10 double mutants, as well as by enhanced first whorl carpeloidy in double mutants of roxy1 with repressors of AG, such as ap2 or lug. Glutaredoxins are oxidoreductases that oxidize or reduce conserved cysteine-containing motifs. Mutagenesis of conserved cysteines within the ROXY1 protein demonstrates the importance of cysteine 49 for its function. Our data demonstrate that, unexpectedly, a plant glutaredoxin is involved in flower development, probably by mediating post-translational modifications of target proteins required for normal petal organ initiation and morphogenesis.

Background The Arabidopsis SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor SPL7 reprograms cellular gene expression to adapt plant growth and cellular metabolism to copper (Cu) limited culture conditions. Plant cells require Cu to maintain essential processes, such as photosynthesis, scavenging reactive oxygen species, cell wall lignification and hormone sensing. More specifically, SPL7 activity promotes a high-affinity Cu-uptake system and optimizes Cu (re-)distribution to essential Cu-proteins by means of specific miRNAs targeting mRNA transcripts for those dispensable. However, the functional mechanism underlying SPL7 activation is still to be elucidated. As SPL7 transcript levels are largely non-responsive to Cu availability, post-translational modification seems an obvious possibility. Previously, it was reported that the SPL7 SBP domain does not bind to DNA in vitro in the presence of Cu ions and that SPL7 interacts with a kin17 domain protein to raise SPL7-target gene expression upon Cu deprivation. Here we report how additional conserved SPL7 protein domains may contribute to the Cu deficiency response in Arabidopsis. Results Cytological and biochemical approaches confirmed an operative transmembrane domain (TMD) and uncovered a dual localisation of SPL7 between the nucleus and an endomembrane system, most likely the endoplasmic reticulum (ER). This new perspective unveiled a possible link between Cu deficit and ER stress, a metabolic dysfunction found capable of inducing SPL7 targets in an SPL7-dependent manner. Moreover, in vivo protein-protein interaction assays revealed that SPL7 is able to homodimerize, probably mediated by the IRPGC domain. These observations, in combination with the constitutive activation of SPL7 targets, when ectopically expressing the N-terminal part of SPL7 including the SBP domain, shed some light on the mechanisms governing SPL7 function. Conclusions Here, we propose a revised model of SPL7 activation and regulation. According to our results, SPL7 would be initially located to endomembranes and activated during ER stress as a result of Cu deficiency. Furthermore, we added the SPL7 dimerization in the presence of Cu ions as an additional regulatory mechanism to modulate the Cu deficiency response. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0231-5) contains supplementary material, which is available to authorized users.

Proper copper (Cu) homeostasis is required by living organisms to maintain essential cellular functions. In the model plant Arabidopsis (Arabidopsis thaliana), the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 (SPL7) transcription factor participates in reprogramming global gene expression during Cu insufficiency in order to improve the metal uptake and prioritize its distribution to Cu proteins of major importance. As a consequence, spl7 null mutants show morphological and physiological disorders during Cu-limited growth, resulting in lower fresh weight, reduced root elongation, and chlorosis. On the other hand, the Arabidopsis KIN17 homolog belongs to a well-conserved family of essential eukaryotic nuclear proteins known to be stress activated and involved in DNA and possibly RNA metabolism in mammals. In the study presented here, we uncovered that Arabidopsis KIN17 participates in promoting the Cu deficiency response by means of a direct interaction with SPL7. Moreover, the double mutant kin17-1 spl7-2 displays an enhanced Cu-dependent phenotype involving growth arrest, oxidative stress, floral bud abortion, and pollen inviability. Taken together, the data presented here provide evidence for SPL7 and KIN17 protein interaction as a point of convergence in response to both Cu deficiency and oxidative stress.

The substructure of pollen exine inCedrus deodara (Roxb.) Loud. andMetasequoia glyptostroboides Hu et Cheng has been examined with an atomic force microscope (AFM). The results indicate that the exine substructure units containing sporopollenin in two species are similar in shape, which are granular, but slightly different in size. InCedrus the substructure unit of pollen exine appears to be 56–99 nm long and 42–74 nm wide, while inMetasequoia it appears to be 81–118 nm long and 43–98 nm wide. It has been observed that the subunits of pollen exine inCedrus arranged tightly to form short-rod-like or spheroidal pollen exine units, several or more than ten of which formed an island-like structure. There are various spaces among these island-like structures which are interconnected to occupy the entire pollen exine. InMetasequoia, the subunits of pollen exine also arranged tightly with a distribution tendency of cluster of 3–10, however, no obvious boundary exists among these clusters. From our results, it is concluded that there is no tendency of helical arrangement for the subunits of pollen exine inCedrus andMetasequoia, and the results support Southworth’ view that subunits of pollen exine are granular shape in lattice structure.

SPL8 and miR156-targeted SPL genes are known to play an essential role in Arabidopsis anther development. Here we show that these SPL genes are also expressed within the developing gynoecium, where they redundantly control development of the female reproductive tract. Whereas the gynoecium morphology in the spl8 single mutant is largely normal, additional down-regulation of miR156-targeted SPL genes results in a shortened style and an apically swollen ovary narrowing onto an elongated gynophore. In particular, the septum does not form properly and lacks a transmitting tract. Loss of SPL8 function enhances the mutant phenotypes of ett, crc and spt, indicating a functional overlap between SPL8 and these genes in regulating gynoecium development. Furthermore, gynoecium development of 35S:MIR156b spl8-1 double mutants shows enhanced sensitivity to a polar auxin transport inhibitor, and the expression pattern of the auxin biosynthesis gene YUCCA4 is altered compared to wild-type. Our observations imply that SPL8 and miR156-targeted SPL genes control gynoecium patterning through interference with auxin homeostasis and signalling.

The non-miR156 targeted SBP-box gene SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8 (SPL8), plays an important role in Arabidopsis anther development, where its loss-of-function results in a semi-sterile phenotype. Fully male-sterile plants are obtained when a spl8 loss-of-function mutation is introduced into a 35S:MIR156 genetic background, thereby revealing functional redundancy between SPL8 and miR156-targeted SBP-box genes. Here, we show that BIM1, a gene encoding a bHLH protein involved in brassinosteroid signaling and embryonic patterning, functions redundantly with SPL8 in its requirement for male fertility. Although bim1 single mutants displayed a mild fertility problem due to shortened filaments in some flowers, mutation of BIM1 significantly enhanced the semi-sterile phenotype of the spl8 mutant. Expression of both SPL8 and BIM1 was detected in overlapping expression domains during early anther developmental stages. Our data suggest that in regulating anther development, SPL8 and BIM1 function cooperatively in a common complex or in synergistic pathways. Phylogenetic analysis supports the idea of an evolutionary conserved function for both genes in angiosperm anther development.

SBP-box genes represent transcription factors ubiquitously found in the plant kingdom and recognized as important regulators of many different aspects of plant development. In this study, 15 SBP-box gene family members were identified in tomato and analyzed with respect to their genomic organization and other structural features. Phylogenetic reconstruction based on the DNA-binding SBP-domain, allowed the classification of the SlySBP proteins into eight groups representing clear orthologous relationships to family members of other flowering plants and the moss Physcomitrella. In order to have a better understanding of their possible function in the development of a fleshy-fruit species like tomato, the mRNA expression levels of all SlySBP genes were quantified in vegetative and reproductive organs of plants, at different stages of growth. As transcripts of ten SlySBP genes were found to carry putative miR156- and miR157-response elements, the expression levels of the corresponding microRNAs were determined as well, revealing different patterns of expression. In addition, eight putative miR156 and four miR157 encoding loci could be identified in the tomato genome, four of them forming a polycistronic cluster. Whereas miR156 and miR157 levels were highest in seedlings, leaves and anthers of young flowers, most miR156-targeted SlySBP genes were found to be expressed in young inflorescences and during fruit development and ripening, suggesting a particularly important role during tomato reproductive growth. The data presented provide a basis for future clarification of the various functions that SBP-box gene family members play in tomato growth and development.

Anther development is an important process for the successful sexual reproduction in plants. Whereas the regulation of the late stages of anther development is quite well described in A. thaliana, little is known about the regulation of the early stages of this process. Two novel groups of factors involved in these early stages have recently been described, namely ROXYs, members of the Glutaredoxin (GRX) family of small and ubiquitous oxidoreductases involved in various cellular and stress-related responses, and SBP-box Genes. ROXYs belong to the CC-type of GRXs with a CCXC active motif and are specific for higher plants. SBP-box genes encode for SQUAMOSA PROMOTER BINDING PROTEIN transcription factors, many of which are targeted by miR156 and miR157. Strikingly, both the enzymes and the transcription factors represent evolutionary conserved gene families and loss-of-function of these genes exhibits similar anther phenotypes, e.g. arresting sporogenous cell formation and missing pollen sacs. This mini-review gives an overview of how these factors affect early anther development and discusses a possible relationship between these factors and other known early anther genes.

The SBP-box transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE8 (SPL8) is required for proper development of sporogenic tissues in Arabidopsis thaliana. Here, we show that the semisterile phenotype of SPL8 loss-of-function mutants is due to partial functional redundancy with several other members of the Arabidopsis SPL gene family. In contrast with SPL8, the transcripts of these latter SPL genes are all targeted by miR156/7. Whereas the introduction of single miR156/7-resistant SPL transgenes could only partially restore spl8 mutant fertility, constitutive overexpression of miR156 in an spl8 mutant background resulted in fully sterile plants. Histological analysis of the anthers of such sterile plants revealed an almost complete absence of sporogenous and anther wall tissue differentiation, a phenotype similar to that reported for sporocyteless/nozzle (spl/nzz) mutant anthers. Expression studies indicated a functional requirement for miR156/7-targeted SPL genes limited to early anther development. Accordingly, several miR156/7-encoding loci were found expressed in anther tissues at later stages of development. We conclude that fully fertile Arabidopsis flowers require the action of multiple miR156/7-targeted SPL genes in concert with SPL8. Either together with SPL/NZZ or independently, these SPL genes act to regulate genes mediating cell division, differentiation, and specification early in anther development. Furthermore, SPL8 in particular may be required to secure fertility of the very first flowers when floral transition-related miR156/7 levels might not have sufficiently declined.

Glutaredoxins (GRXs) are small oxidoreductases that are involved in various cellular processes and play a crucial role in responses to oxidative stress. Three GRX subgroups exist in plants, and GRXs with active sites of the CPYC and CGFS types are common to pro- and eukaryotes. In contrast, GRXs with the CC type motif have so far only been identified in land plants. Here, we report that the two CC-type GRXs ROXY1 and ROXY2 together control anther development in Arabidopsis thaliana. Single roxy1 and roxy2 mutants are fertile and produce normal anthers. However, roxy1 roxy2 double mutants are sterile and do not produce pollen. Strikingly, abaxial and adaxial anther lobe differentiation are differently affected, with early lobe differentiation being defective in the adaxial lobes, whereas later steps during pollen mother cell differentiation are disrupted in the abaxial lobes. Expression studies show that ROXY1 and ROXY2 are expressed with overlapping patterns during anther development. Lack of ROXY1 and ROXY2 function affects a large number of anther genes at the transcriptional level. Genetic and RT-PCR data imply that ROXY1/2 function downstream of the early-acting anther gene SPOROCYTELESS/NOZZLE and upstream of DYSFUNCTIONAL TAPETUM1, controlling tapetum development. Mutagenesis of a conserved glutathione-binding glycine in the ROXY1 protein indicates that CC-type GRXs need to interact with glutathione to catalyze essential biosynthetic reactions. Analysis of these two novel anther genes indicates that redox regulation, as well as participating in plant stress defense mechanisms, might play a major role in the control of male gametogenesis.

To study gene function, generation of loss-of-function mutants by RNA interference (RNAi) approaches and T-DNA insertional mutagenesis are widely used in plant science and have proven to be very successful in determining gene functions. AGAMOUS-LIKE18 ( AGL18 ) is a MADS-box gene that is expressed

Glutaredoxins (GRXs) are small, ubiquitous oxidoreductases that have been intensively studied in E. coli, yeast and humans. They are involved in a large variety of cellular processes and exert a crucial function in the response to oxidative stress. GRXs can reduce disulfides by way of conserved cysteines, located in conserved active site motifs. As in E. coli, yeast, and humans, GRXs with active sites of the CPYC and CGFS type are also found in lower and higher plants, however, little has been known about their function. Surprisingly, 21 GRXs from Arabidopsis thaliana contain a novel, plant-specific CC type motif. Lately, information on the function of CC type GRXs and redox regulation, in general, is accumulating. This review focuses on recent findings indicating that GRXs, glutathione and redox regulation, in general, seem to be involved in different processes of development, so far, namely in the formation of the flower. Recent advances in EST and genome sequencing projects allowed searching for the presence of the three different types of the GRX subclasses in other evolutionary informative plant species. A comparison of the GRX subclass composition from Physcomitrella, Pinus, Oryza, Populus, and Arabidopsis is presented. This analysis revealed that only two CC type GRXs exist in the bryophyte Physcomitrella and that the CC type GRXs group expanded during the evolution of land plants. The existence of a large CC type subclass in angiosperms supports the assumption that their capability to modify target protein activity posttranslationally has been integrated into crucial plant specific processes involved in higher plant development.

Proteins harboring the kin17 domain (KIN17) constitute a family of well-conserved eukaryotic nuclear proteins involved in nucleic acid metabolism. In mammals, KIN17 orthologs contribute to DNA replication, RNA splicing, and DNA integrity maintenance. Recently, we reported a functional characterization of an Arabidopsis thaliana KIN17 homolog (AtKIN17) that uncovered a role for this protein in tuning physiological responses during copper (Cu) deficiency and oxidative stress. However, functions similar to those described in mammals may also be expected in plants given the conservation of functional domains in KIN17 orthologs. Here, we provide additional data consistent with the participation of AtKIN17 in controlling general plant growth and development, as well as in response to UV radiation. Furthermore, the Arabidopsis genome codes for a second homolog to KIN17, we referred to as KIN17-like-protein (KLP). KLP loss-of-function lines exhibited a reduced inhibition of root growth in response to copper excess and relatively elongated hypocotyls in etiolated seedlings. Altogether, our experimental data point to a general function of the kin17 domain proteins in plant growth and development.