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Abstract Background Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species. Results We developed a simple and efficient method to isolate small RNAs from different plant species by first comparing different total RNA extraction protocols, followed by streamlining the best one, finally resulting in a small RNA extraction method that has no need of first total RNA extraction and is not based on the commercially available TRIzol® Reagent or columns. This small RNA extraction method not only works well for plant tissues with high polysaccharide content, like cactus, agave, banana, and tomato, but also for plant species like Arabidopsis or tobacco. Furthermore, the obtained small RNA samples were successfully used in northern blot assays. Conclusion Here we provide a simple and efficient method to isolate small RNAs from different plant species, such as cactus, agave, banana, tomato, Arabidopsis, and tobacco, and the small RNAs from this simplified and low cost method is suitable for downstream handling like northern blot assays.

The bHLH transcription factor SPATULA (SPT) has been identified as a regulator during different stages of Arabidopsis development, including the control of leaf size. However, the mechanism via which it performs this function has not been elucidated. To better understand the role of SPT during leaf development, we used a transcriptomic approach to identify putative target genes. We found putative SPT target genes related to leaf development, and to stomata and trichome formation. Furthermore, genes related to anthocyanin biosynthesis. In this work, we demonstrate that SPT is a negative regulator of stomata number and a positive regulator of trichome number. In addition, SPT is required for sucrose-mediated anthocyanin biosynthesis.

The WIP family of transcription factors comprises the A1d subgroup of C2H2 zinc finger proteins. This family has six members in Arabidopsis thaliana and most of the known functions have been described by analyzing single knockout mutants. However, it has been shown that WIP2 and its closest paralogs WIP4 and WIP5 have a redundant and essential function in root meristems. It is likely that these and other WIP genes perform more, still unknown, functions. To obtain hints about these other functions, the expression of the six WIP genes was explored. Moreover, phenotypic ana-lyses of overexpressors and wip mutants revealed functions in modulating organ and cell size, stomatal density, and vasculature development.

The gynoecium is the most complex organ formed by the flowering plants. It encloses the ovules, provides a surface for pollen contact and self-incompatibility reactions, allows pollen tube growth, and, post fertilization, develops into the fruit. Consequently, the regulation of gynoecium morphogenesis is complex and appropriate timing of this process in part determines reproductive success. However, little is known about the global control of gynoecium development, even though many regulatory genes have been characterized. Here, we characterized dynamic gene expression changes using laser-microdissected gynoecium tissue from four developmental stages in Arabidopsis. We provide a high-resolution map of global expression dynamics during gynoecium morphogenesis and link these to the gynoecium interactome. We reveal groups of genes acting together early and others acting late in morphogenesis. Clustering of co-expressed genes enables comparisons between the leaf, shoot apex, and gynoecium transcriptomes, allowing the dissection of common and distinct regulators. Furthermore, our results lead to the discovery of genes with putative transcription factor activity (B3LF1, -2, DOFLF1), which, when mutated, lead to impaired gynoecium expansion, illustrating that global transcriptome analyses reveal yet unknown developmental regulators. Our data show that genes encoding highly interacting proteins, such as SEPALLATA3, AGAMOUS, and TOPLESS, are expressed evenly during development but switch interactors over time, whereas stage-specific proteins tend to have fewer interactors. Our analysis connects specific transcriptional regulator activities, protein interactions, and underlying metabolic processes, contributing toward a dynamic network model for gynoecium development., (© American Society of Plant Biologists 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The phytohormone cytokinin is crucial for plant growth and development. The site of action of cytokinin in the plant is dependent on the expression of the cytokinin receptors. In Arabidopsis, there are three cytokinin receptors that present some overlap in expression pattern. Functional studies demonstrated that the receptors play highly redundant roles but also have specialized functions. Here, we focus on gynoecium development, which is the female reproductive part of the plant. Cytokinin signaling has been demonstrated to be important for reproductive development, positively affecting seed yield and fruit production. Most of these developmental processes are regulated by cytokinin during early gynoecium development. While some information is available, there is a gap in knowledge on cytokinin function and especially on the cytokinin receptors during early gynoecium development. Therefore, we studied the expression patterns and the role of the cytokinin receptors during gynoecium development. We found that the three receptors are expressed in the gynoecium and that they have redundant and specialized functions., (Copyright © 2020 Cerbantez-Bueno, Zúñiga-Mayo, Reyes-Olalde, Lozano-Sotomayor, Herrera-Ubaldo, Marsch-Martinez and de Folter.)

Background: Alkamides are plant-specific bioactive molecules. They are low molecular weight N-substituted α-unsaturated acyl amides that display biological explicit activities in different organisms from bacteria, fungi, insects to mammals and plants. The acyl chain has been proposed to be biosynthesized from a fatty acid; however, this has not been demonstrated yet. Heliopsis longipes (Asteraceae) accumulates in root a C10 alkamide called affinin in its roots, but not in leaves. The closely related species Heliopsis annua does not produce alkamides. To elucidate the biosynthetic pathway of the alkamides acyl chain, a comparative global gene expression analysis contrasting roots and leaves of both species was performed., Methods: Transcriptomics analysis allowed to identify genes highly expressed in H. longipes roots, but not in tissues and species that do not accumulate alkamides. The first domain searched was the Ketosynthase (KS) domain. The phylogenetic analysis using sequences of the KS domain of FAS and PKS from different organisms, revealed that KS domains of the differentially expressed transcripts in H. longipes roots and the KS domain found in transcripts of Echinacea purpurea , another alkamides producer species, were grouped together with a high bootstrap value of 100%, sharing great similarity. Among the annotated transcripts, we found some coding for the enzymatic domains KS, AT, ACP, DH, OR and TE, which presented higher expression in H. longipes roots than in leaves. The expression level of these genes was further evaluated by qRT-PCR. All unigenes tested showed higher expression in H. longipes roots than in any the other samples. Based on this and considering that the acyl chain of affinin presents unsaturated bonds at even C numbers, we propose a new putative biosynthesis pathway mediated by a four modules polyketide synthase (PKS)., Results: The global gene expression analysis led to the selection of a set of candidate genes involved in the biosynthesis of the acyl chain of affinin, suggesting that it may be performed by a non-iterative, partially reductive, four module type I PKS complex (PKS alk ) previously thought to be absent from the plant kingdom., Competing Interests: The authors declare that they have no competing interests., (© 2020 Buitimea-Cantúa et al.)

The apical-basal axis of the Arabidopsis gynoecium is established early during development and is divided into four elements from the bottom to the top: the gynophore, the ovary, the style, and the stigma. Currently, it is proposed that the hormone auxin plays a critical role in the correct apical-basal patterning through a concentration gradient from the apical to the basal part of the gynoecium, as chemical inhibition of polar auxin transport through 1-N-naphtylphtalamic acid (NPA) application, severely affects the apical-basal patterning of the gynoecium. In this work, we show that the apical-basal patterning of gynoecia is also sensitive to exogenous cytokinin (benzyl amino purine, BAP) application in a similar way as to NPA. BAP and NPA treatments were performed in different mutant backgrounds where either cytokinin perception or auxin transport and perception were affected. We observed that cytokinin and auxin signaling mutants are hypersensitive to NPA treatment, and auxin transport and signaling mutants are hypersensitive to BAP treatment. BAP effects in apical-basal gynoecium patterning are very similar to the effects of NPA, therefore, it is possible that BAP affects auxin transport in the gynoecium. Indeed, not only the cytokinin-response TCS::GFP marker, but also the auxin efflux carrier PIN1 (PIN1::PIN1:GFP) were both affected in BAP-induced valveless gynoecia, suggesting that the BAP treatment producing the morphological changes has an impact on both in the response pattern to cytokinin and on auxin transport. In summary, we show that cytokinin affects proper apical-basal gynoecium patterning in Arabidopsis in a similar way to the inhibition of polar auxin transport, and that auxin and cytokinin mutants and markers suggest a relation between both hormones in this process.

Freshwater is a limited and dwindling global resource; therefore, efficient water use is required for food crops that have high water demands, such as rice, or for the production of sustainable energy biomass. We show here that expression of the Arabidopsis HARDY (HRD) gene in rice improves water use efficiency, the ratio of biomass produced to the water used, by enhancing photosynthetic assimilation and reducing transpiration. These drought-tolerant, low-water-consuming rice plants exhibit increased shoot biomass under well irrigated conditions and an adaptive increase in root biomass under drought stress. The HRD gene, an AP2/ERF-like transcription factor, identified by a gain-of-function Arabidopsis mutant hrd-D having roots with enhanced strength, branching, and cortical cells, exhibits drought resistance and salt tolerance, accompanied by an enhancement in the expression of abiotic stress associated genes. HRD overexpression in Arabidopsis produces thicker leaves with more chloroplast-bearing mesophyll cells, and in rice, there is an increase in leaf biomass and bundle sheath cells that probably contributes to the enhanced photosynthesis assimilation and efficiency. The results exemplify application of a gene identified from the model plant Arabidopsis for the improvement of water use efficiency coincident with drought resistance in the crop plant rice.

The BOLITA (BOL) gene, an AP2/ERF transcription factor, was characterized with the help of an activation tag mutant and overexpression lines in Arabidopsis and tobacco. The leaf size of plants overexpressing BOL was smaller than wild type plants due to a reduction in both cell size and cell number. Moreover, severe overexpressors showed ectopic callus formation in roots. Accordingly, global gene expression analysis using the overexpression mutant reflected the alterations in cell proliferation, differentiation and growth through expression changes in RBR, CYCD, and TCP genes, as well as genes involved in cell expansion (i.e. expansins and the actin remodeling factor ADF5). Furthermore, the expression of hormone signaling (i.e. auxin and cytokinin), biosynthesis (i.e. ethylene and jasmonic acid) and regulatory genes was found to be perturbed in bol-D mutant leaves.

The formation and the development of the floral organs require an intercalate expression of organ-specific genes. At the same time, meristem-specific genes are repressed to complete the differentiation of the organs in the floral whorls. In an Arabidopsis activation tagging population, a mutant affected in inflorescence architecture was identified. This gain-of-function mutant, designated downwards siliques1 (dsl1-D), has shorter internodes and the lateral organs such as flowers are bending downwards, similar to the loss-of-function brevipedicellus (bp) mutant. The affected gene in dsl1-D appeared to be ASYMMETRIC LEAVES2-LIKE1 (ASL1)/LATERAL ORGAN BOUNDARIES domain gene 36 (LBD36), which is a member of the ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) domain gene family. Analysis of the loss-of-function mutant asl1/lbd36 did not show morphological aberration. Double mutant analysis of asl1/lbd36 together with as2, the ASL1/LBD36 closest homologue, demonstrates that these two members of the AS2/LOB family act partially redundant to control cell fate determination in Arabidopsis petals. Moreover, molecular analysis revealed that overexpression of ASL1/LBD36 leads to repression of the homeobox gene BP, which supports the model that an antagonistic relationship between ASL/LBD and homeobox members is required for the differentiation of lateral organs.

A method for the generation of stable activation tag inserts was developed in Arabidopsis using the maize (Zea mays) En-I transposon system. The method employs greenhouse selectable marker genes that are useful to efficiently generate large populations of insertions. A population of about 8,300 independent stable activation tag inserts has been produced. Greenhouse-based screens for mutants in a group of plants containing about 2,900 insertions revealed about 31 dominant mutants, suggesting a dominant mutant frequency of about 1%. From the first batch of about 400 stable insertions screened in the greenhouse, four gain-in-function, dominant activation-tagged, morphological mutants were identified. A novel gain-in-function mutant called thread is described, in which the target gene belongs to the same family as the YUCCA flavin-mono-oxygenase that was identified by T-DNA activation tagging. The high frequency of identified gain-in-function mutants in the population suggests that the En-I system described here is an efficient strategy to saturate plant genomes with activation tag inserts. Because only a small number of primary transformants are required to generate an activation tag population, the En-I system appears to be an attractive alternative to study plant species where the present transformation methods have low efficiencies.