The Arabidopsis ATP-Binding Cassette E protein ABCE2 is a conserved component of the translation machinery.

ATP-Binding Cassette E (ABCE) proteins dissociate cytoplasmic ribosomes after translation terminates, and contribute to ribosome recycling, thus linking translation termination to initiation. This function has been demonstrated to be essential in animals, fungi, and archaea, but remains unexplored in plants. In most species, ABCE is encoded by a single-copy gene; by contrast, Arabidopsis thaliana has two ABCE paralogs, of which ABCE2 seems to conserve the ancestral function. We isolated apiculata7-1 (api7-1), the first viable, hypomorphic allele of ABCE2, which has a pleiotropic morphological phenotype reminiscent of mutations affecting ribosome biogenesis factors and ribosomal proteins. We also studied api7-2, a null, recessive lethal allele of ABCE2. Co-immunoprecipitation experiments showed that ABCE2 physically interacts with components of the translation machinery. An RNA-seq study of the api7-1 mutant showed increased responses to iron and sulfur starvation. We also found increased transcript levels of genes related to auxin signaling and metabolism. Our results support for the first time a conserved role for ABCE proteins in translation in plants, as previously shown for the animal, fungal, and archaeal lineages. In Arabidopsis, the ABCE2 protein seems important for general growth and vascular development, likely due to an indirect effect through auxin metabolism.

Dissection of the Arabidopsis HUA-PEP gene activity reveals that ovule fate specification requires restriction of the floral A-function.

Ovules are essential for sexual plant reproduction and seed formation, and are fundamental for agriculture. However, our understanding of the molecular mechanisms governing ovule development is far from complete. In Arabidopsis, ovule identity is determined by homeotic MADS-domain proteins that define the floral C- (AG) and D- (SHP1/SHP2, STK) functions. Pre-mRNA processing of these genes is critical and mediated by HUA-PEP activity, composed of genes encoding RNA-binding proteins. In strong hua-pep mutants, functional transcripts for C- and D-function genes are reduced, resulting in homeotic transformation of ovules. Thus, hua-pep mutants provide an unique sensitized background to study ovule morphogenesis when C- and D-functions are simultaneously compromised. We found that hua-pep ovules are morphologically sepaloid and show ectopic expression of the homeotic class-A gene AP1. Inactivation of AP1 or AP2 (A-function genes) in hua-pep mutants reduced homeotic conversions, rescuing ovule identity while promoting carpelloid traits in transformed ovules. Interestingly, increased AG dosage led to similar results. Our findings strongly suggest that HUA-PEP activity is required for correct C and D floral functions, which in turn prevents ectopic expression of class-A genes in ovules for their proper morphogenesis, evoking the classic A-C antagonism of the ABC model for floral organ development.

Arabidopsis HUA ENHANCER 4 delays flowering by upregulating the MADS-box repressor genes FLC and MAF4.

The adaptive success of flowering plants is largely due to their ability to align floral production with optimal conditions. In Arabidopsis thaliana, MADS-box repressors of the FLC/MAF-clade prevent flowering under non-inductive conditions, although the role of some members is not yet clearly defined. Using a genetic strategy, we identified the KH-domain gene HEN4, previously shown to be involved in MADS-box floral homeotic gene regulation, as a modulator of flowering time. Loss-of-function hen4 mutants are early-flowering, and their response to low growth-temperature (16 °C) and day-length is altered. Interestingly, hen4 plants showed dramatic reduction of FLC and MAF4 transcripts, whereas other flowering repressors of the same clade (FLM, MAF2, MAF3, MAF5) remained unaltered. We also determined that hen4, partly due to loss of FLC, accelerates the vegetative phase-change. This report provides insight into flowering time control and highlights the potential of versatile regulators such as HEN4 to coordinate the juvenile-to-adult transition and floral timing.

Ovule identity mediated by pre-mRNA processing in Arabidopsis.

Ovules are fundamental for plant reproduction and crop yield as they are the precursors of seeds. Therefore, ovule specification is a critical developmental program. In Arabidopsis thaliana, ovule identity is redundantly conferred by the homeotic D-class genes SHATTERPROOF1 (SHP1), SHP2 and SEEDSTICK (STK), phylogenetically related to the MADS-domain regulatory gene AGAMOUS (AG), essential in floral organ specification. Previous studies have shown that the HUA-PEP activity, comprised of a suite of RNA-binding protein (RBP) encoding genes, regulates AG pre-mRNA processing and thus flower patterning and organ identity. Here, we report that the HUA-PEP activity additionally governs ovule morphogenesis. Accordingly, in severe hua-pep backgrounds ovules transform into flower organ-like structures. These homeotic transformations are most likely due to the dramatic reduction in SHP1, SHP2 and STK activity. Our molecular and genome-wide profiling strategies revealed the accumulation of prematurely terminated transcripts of D-class genes in hua-pep mutants and reduced amounts of their respective functional messengers, which points to pre-mRNA processing misregulation as the origin of the ovule developmental defects in such backgrounds. RNA processing and transcription are coordinated by the RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD). Our results show that HUA-PEP activity members can interact with the CTD regulator C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 (CPL1), supporting a co-transcriptional mode of action for the HUA-PEP activity. Our findings expand the portfolio of reproductive developmental programs in which HUA-PEP activity participates, and further substantiates the importance of RNA regulatory mechanisms (pre-mRNA co-transcriptional regulation) for correct gene expression during plant morphogenesis.

Arabidopsis thaliana root colonization by the nematophagous fungus Pochonia chlamydosporia is modulated by jasmonate signaling and leads to accelerated flowering and improved yield.

Pochonia chlamydosporia has been intensively studied in nematode control of different crops. We have investigated the interaction between P. chlamydosporia and the model system Arabidopsis thaliana under laboratory conditions in the absence of nematodes. This study demonstrates that P. chlamydosporia colonizes A. thaliana. Root colonization monitored with green fluorescent protein-tagged P. chlamydosporia and quantitative PCR (qPCR) quantitation methods revealed root cell invasion. Fungal inoculation reduced flowering time and stimulated plant growth, as determined by total FW increase, faster development of inflorescences and siliques, and a higher yield in terms of seed production per plant. Precocious flowering was associated with significant expression changes in key flowering-time genes. In addition, we also provided molecular and genetic evidence that point towards jasmonate signaling as an important factor to modulate progression of plant colonization by the fungus. Our results indicate that P. chlamydosporia provides benefits to the plant in addition to its nematophagous activity. This report highlights the potential of P. chlamydosporia to improve yield in economically important crops.

K-homology nuclear ribonucleoproteins regulate floral organ identity and determinacy in arabidopsis.

Post-transcriptional control is nowadays considered a main checking point for correct gene regulation during development, and RNA binding proteins actively participate in this process. Arabidopsis thaliana FLOWERING LOCUS WITH KH DOMAINS (FLK) and PEPPER (PEP) genes encode RNA-binding proteins that contain three K-homology (KH)-domain, the typical configuration of Poly(C)-binding ribonucleoproteins (PCBPs). We previously demonstrated that FLK and PEP interact to regulate FLOWERING LOCUS C (FLC), a central repressor of flowering time. Now we show that FLK and PEP also play an important role in the maintenance of the C-function during floral organ identity by post-transcriptionally regulating the MADS-box floral homeotic gene AGAMOUS (AG). Previous studies have indicated that the KH-domain containing protein HEN4, in concert with the CCCH-type RNA binding protein HUA1 and the RPR-type protein HUA2, facilitates maturation of the AG pre-mRNA. In this report we show that FLK and PEP genetically interact with HEN4, HUA1, and HUA2, and that the FLK and PEP proteins physically associate with HUA1 and HEN4. Taken together, these data suggest that HUA1, HEN4, PEP and FLK are components of the same post-transcriptional regulatory module that ensures normal processing of the AG pre-mRNA. Our data better delineates the roles of PEP in plant development and, for the first time, links FLK to a morphogenetic process.

3' Rapid Amplification of cDNA Ends (3' RACE) Using Arabidopsis Samples.

Production of functional eukaryotic RNA is a very elaborate process that involves a complex interplay between transcription and various RNA processing activities, including splicing, 5' capping, and 3' cleavage and polyadenylation (Bentley, 2014). Accurate mapping of RNA ends provides a valuable tool to assess transcriptional and post-transcriptional events giving rise to different gene transcripts. The abundance of such transcripts most likely depends on exogenous and developmental cues, or mutations. In the reference plant Arabidopsis, perturbation of the HUA-PEP post-transcriptional regulatory factors (Rodríguez-Cazorla et al., 2015) leads to the accumulation of aberrant transcripts of the key floral homeotic gene AGAMOUS (AG) (Yanofsky et al., 1990) that retain intronic sequence. It was determined by 3' RACE reactions that such erroneous transcripts correspond to premature processing and polyadenylation events taking place at the AG intron region. Here we describe a protocol that is suitable for analysis of relatively abundant transcripts and also for detecting aberrant RNA species that are likely prone to rapid turnover. Likewise, the method, here adapted to Arabidopsis reproductive tissues, can be applied to characterize RNA species from other organs (leaf, root) and/or other plant species. We provide a detailed protocol of our 3' RACE procedure comprising four major parts: Total RNA extraction, RNA amount determination and quality control, the RACE procedure itself, and isolation of the resulting RACE products for cloning and sequencing.

Antagonistic gene activities determine the formation of pattern elements along the mediolateral axis of the Arabidopsis fruit.

The Arabidopsis fruit mainly consists of a mature ovary that shows three well defined territories that are pattern elements along the mediolateral axis: the replum, located at the medial plane of the flower, and the valve and the valve margin, both of lateral nature. JAG/FIL activity, which includes the combined functions of JAGGED (JAG), FILAMENTOUS FLOWER (FIL), and YABBY3 (YAB3), contributes to the formation of the two lateral pattern elements, whereas the cooperating genes BREVIPEDICELLUS (BP) and REPLUMLESS (RPL) promote replum development. A recent model to explain pattern formation along the mediolateral axis hypothesizes that JAG/FIL activity and BP/RPL function as antagonistic lateral and medial factors, respectively, which tend to repress each other. In this work, we demonstrate the existence of mutual exclusion mechanisms between both kinds of factors, and how this determines the formation and size of the three territories. Medial factors autonomously constrain lateral factors so that they only express outside the replum, and lateral factors negatively regulate the medially expressed BP gene in a non-autonomous fashion to ensure correct replum development. We also have found that ASYMMETRIC LEAVES1 (AS1), previously shown to repress BP both in leaves and ovaries, collaborates with JAG/FIL activity, preventing its repression by BP and showing synergistic interactions with JAG/FIL activity genes. Therefore AS gene function (the function of the interacting genes AS1 and AS2) has been incorporated in the model as a new lateral factor. Our model of antagonistic factors provides explanation for mutant fruit phenotypes in Arabidopsis and also may help to understand natural variation of fruit shape in Brassicaceae and other species, since subtle changes in gene expression may cause conspicuous changes in the size of the different tissue types.

Antagonistic interactions between Arabidopsis K-homology domain genes uncover PEPPER as a positive regulator of the central floral repressor FLOWERING LOCUS C.

Plant floral transition is a major developmental switch regulated by an integrated network of pathways. Arabidopsis FLOWERING LOCUS K (FLK), a protein with three KH RNA-binding domains, operates in the autonomous flowering-promotive pathway by decreasing the transcript levels of the key flowering repressor FLOWERING LOCUS C (FLC). Here we report that PEPPER (PEP), an FLK paralog previously shown to affect vegetative and pistil development, antagonizes FLK by positively regulating FLC. Lack of PEP function rescues the flk late-flowering phenotype with a concomitant decrease in FLC RNA levels. Loss of HUA2, another FLC activator encoding an RNA-binding protein, further rescues flk, being flk hua2 pep triple mutants virtually wild-type regarding flowering time. Consistently, PEP overexpression determines high levels of FLC transcripts and flowering delay. Genetic and molecular analyses indicate that FLK and PEP act independently of FCA, another important FLC repressor in the autonomous pathway. In addition, we present data suggesting that PEP may affect FLC expression at both transcriptional and post-transcriptional levels. Overall, our results uncover PEP as a new factor for FLC upregulation, underscoring the importance of RNA-binding activities during developmental timing of flowering.

Alteration of the shoot radial pattern in Arabidopsis thaliana by a gain-of-function allele of the class III HD-Zip gene INCURVATA4.

Class III HD-Zip (HD-Zip III) family genes play key roles in a number of fundamental developmental programs in Arabidopsis thaliana, such as embryo patterning, meristem initiation and homeostasis, lateral organ polarity and vascular development. Semidominant gain-of-function alleles of the HD-Zip III genes PHABULOSA (PHB), PHAVOLUTA (PHV) and REVOLUTA (REV) disrupt the negative regulation of these genes by a mechanism of microRNA interference. We provide evidence that the gain-of-function icu4-1 allele of INCURVATA4, a gene encoding the HD-Zip III transcription factor ATHB15/CORONA (CNA), stimulates the production of vascular tissues, supporting a role for ICU4 in promoting vascular development. Occasionally, homozygous mutants for this allele show a reduced number of thick shoot vascular bundles, although normal collateral polarity remains unchanged. Genetic analysis of icu4-1 and phb-1D, a gain-of-function allele of the related PHB gene, revealed antagonism in lateral organ polarity between both mutations and a synergistic interaction in shoots, with transformation of the polarized collateral bundles into a radialized amphivasal pattern. These results indicate that the precise regulation of HD-Zip III genes confers positional information which is required to establish the number and pattern of vascular bundles in the stem. In addition, we present results that suggest an interaction between ICU4 function and auxin signaling.

Common regulatory networks in leaf and fruit patterning revealed by mutations in the Arabidopsis ASYMMETRIC LEAVES1 gene.

Carpels and leaves are evolutionarily related organs, as the former are thought to be modified leaves. Therefore, developmental pathways that play crucial roles in patterning both organs are presumably conserved. In leaf primordia of Arabidopsis thaliana, the ASYMMETRIC LEAVES1 (AS1) gene interacts with AS2 to repress the class I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS (BP), KNAT2 and KNAT6, restricting the expression of these genes to the meristem. In this report, we describe how AS1, presumably in collaboration with AS2, patterns the Arabidopsis gynoecium by repressing BP, which is expressed in the replum and valve margin, interacts in the replum with REPLUMLESS (RPL), an essential gene for replum development, and positively regulates the expression of this gene. Misexpression of BP in the gynoecium causes an increase in replum size, while the valve width is slightly reduced, and enhances the effect of mutations in FRUITFULL (FUL), a gene with an important function in valve development. Altogether, these findings strongly suggest that BP plays a crucial role in replum development. We propose a model for pattern formation along the mediolateral axis of the ovary, whereby three domains (replum, valve margin and valve) are specified by the opposing gradients of two antagonistic factors, valve factors and replum factors, the class I KNOX genes working as the latter.

The HVE/CAND1 gene is required for the early patterning of leaf venation in Arabidopsis.

The hemivenata-1 (hve-1) recessive allele was isolated in a search for natural variations in the leaf venation pattern of Arabidopsis thaliana, where it was seen to cause extremely simple venation in vegetative leaves and cotyledons, increased shoot branching, and reduced root waving and fertility, traits that are reminiscent of some mutants deficient in auxin signaling. Reduced sensitivity to exogenous auxin was found in the hve-1 mutant, which otherwise displayed a wild-type response to auxin transport inhibitors. The HVE gene was positionally cloned and found to encode a CAND1 protein. The hve-1 mutation caused mis-splicing of the transcripts of the HVE/CAND1 gene and a vein phenotype indistinguishable from that of hve-2 and hve-3, two putatively null T-DNA alleles. Inflorescence size and fertility were more affected by hve-2 and hve-3, suggesting that hve-1 is hypomorphic. The simple venation pattern of hve plants seems to arise from an early patterning defect. We found that HVE/CAND1 binds to CULLIN1, and that the venation patterns of axr1 and hve mutants are similar, which suggest that ubiquitin-mediated auxin signaling is required for venation patterning in laminar organs, the only exception being cauline leaves. Our analyses of double mutant and transgenic plants indicated that auxin transport and perception act independently to pattern leaf veins, and that the HVE/CAND1 gene acts upstream of ATHB-8 at least in higher order veins, in a pathway that involves AXR1, but not LOP1, PIN1, CVP1 or CVP2.

Mutations in the microRNA complementarity site of the INCURVATA4 gene perturb meristem function and adaxialize lateral organs in arabidopsis.

Here, we describe how the semidominant, gain-of-function icu4-1 and icu4-2 alleles of the INCURVATA4 (ICU4) gene alter leaf phyllotaxis and cell organization in the root apical meristem, reduce root length, and cause xylem overgrowth in the stem. The ICU4 gene was positionally cloned and found to encode the ATHB15 transcription factor, a class III homeodomain/leucine zipper family member, recently named CORONA. The icu4-1 and icu4-2 alleles bear the same point mutation that affects the microRNA complementarity site of ICU4 and is identical to those of several semidominant alleles of the class III homeodomain/leucine zipper family members PHABULOSA and PHAVOLUTA. The icu4-1 and icu4-2 mutations significantly increase leaf transcript levels of the ICU4 gene. The null hst-1 allele of the HASTY gene, which encodes a nucleocytoplasmic transporter, synergistically interacts with icu4-1, the double mutant displaying partial adaxialization of rosette leaves and carpels. Our results suggest that the ICU4 gene has an adaxializing function and that it is down-regulated by microRNAs that require the HASTY protein for their biogenesis.

PEPPER, a novel K-homology domain gene, regulates vegetative and gynoecium development in Arabidopsis.

Pistil final morphology relies on floral meristem homeostasis, proper organ specification and regional differentiation. These are developmental processes in which sophisticated signaling networks are being uncovered. However, further elements for fine-tuning adjustment still remain to be disclosed. At the molecular level, posttranscriptional modulators may fit such a profile. In this work, we describe the characterization of PEPPER (PEP), a novel Arabidopsis gene encoding a polypeptide with K-homology (KH) RNA-binding modules, which acts on vegetative growth and pistil development. PEP was initially identified as one of the gene functions affected in a complex mutant carrying a chromosomal reorganization, which exhibits aberrant phyllotaxy and small fruits with supernumerary carpels. In contrast, plants carrying single-gene pep null mutations exhibit subtle morphological alterations. Individuals bearing a stronger-than-null allele present a phenotype comprising leaf alterations, phyllotactic errors and sporadic presence of fruits with multiple valves. Accordingly, dynamic PEP expression was detected in all major organs examined. Complementation experiments with a PEP genomic clone confirmed a role for PEP as a regulator in vegetative and reproductive development. Moreover, our genetic studies suggest that PEP interacts with element(s) of the CLAVATA signaling pathway.