Andy Pereira

pBINPLUS: an improved plant transformation vector based on pBIN19

We describe the construction of a new plant transformation vector, pBINPLUS, based on the popular pBIN19 vector. Improvements over pBIN19 include location of the selectable marker gene at the left T-DNA border, a higher copy number inE. coli, and two rare restriction sites around the multiple cloning site for easier cloning and analysis of T-DNA insertions in plant genomes.

The SHINE Clade of AP2 Domain Transcription Factors Activates Wax Biosynthesis, Alters Cuticle Properties, and Confers Drought Tolerance when Overexpressed in Arabidopsis

The interface between plants and the environment plays a dual role as a protective barrier as well as a medium for the exchange of gases, water, and nutrients. The primary aerial plant surfaces are covered by a cuticle, acting as the essential permeability barrier toward the atmosphere. It is a heterogeneous layer composed mainly of lipids, namely cutin and intracuticular wax with epicuticular waxes deposited on the surface. We identified an Arabidopsis thaliana activation tag gain-of-function mutant shine (shn) that displayed a brilliant, shiny green leaf surface with increased cuticular wax compared with the leaves of wild-type plants. The gene responsible for the phenotype encodes one member of a clade of three proteins of undisclosed function, belonging to the plant-specific family of AP2/EREBP transcription factors. Overexpression of all three SHN clade genes conferred a phenotype similar to that of the original shn mutant. Biochemically, such plants were altered in wax composition (very long fatty acid derivatives). Total cuticular wax levels were increased sixfold in shn compared with the wild type, mainly because of a ninefold increase in alkanes that comprised approximately half of the total waxes in the mutant. Chlorophyll leaching assays and fresh weight loss experiments indicated that overexpression of the SHN genes increased cuticle permeability, probably because of changes in its ultrastructure. Likewise, SHN gene overexpression altered leaf and petal epidermal cell structure, trichome number, and branching as well as the stomatal index. Interestingly, SHN overexpressors displayed significant drought tolerance and recovery, probably related to the reduced stomatal density. Expression analysis using promoter-β-glucuronidase fusions of the SHN genes provides evidence for the role of the SHN clade in plant protective layers, such as those formed during abscission, dehiscence, wounding, tissue strengthening, and the cuticle. We propose that these diverse functions are mediated by regulating metabolism of lipid and/or cell wall components.

Molecular and Physiological Analysis of Drought Stress in Arabidopsis Reveals Early Responses Leading to Acclimation in Plant Growth

Plant drought stress response and resistance are complex biological processes that need to be analyzed at a systems level using genomics and physiological approaches to dissect experimental models that address drought stresses encountered by crops in the field. Toward this goal, a controlled, sublethal, moderate drought (mDr) treatment system was developed in Arabidopsis (Arabidopsis thaliana) as a reproducible assay for the dissection of plant responses to drought. The drought assay was validated using Arabidopsis mutants in abscisic acid (ABA) biosynthesis and signaling displaying drought sensitivity and in jasmonate response mutants showing drought resistance, indicating the crucial role of ABA and jasmonate signaling in drought response and acclimation. A comparative transcriptome analysis of soil water deficit drought stress treatments revealed the similarity of early-stage mDr to progressive drought, identifying common and specific stress-responsive genes and their promoter cis-regulatory elements. The dissection of mDr stress responses using a time-course analysis of biochemical, physiological, and molecular processes revealed early accumulation of ABA and induction of associated signaling genes, coinciding with a decrease in stomatal conductance as an early avoidance response to drought stress. This is accompanied by a peak in the expression of expansin genes involved in cell wall expansion, as a preparatory step toward drought acclimation by the adjustment of the cell wall. The time-course analysis of mDr provides a model with three stages of plant responses: an early priming and preconditioning stage, followed by an intermediate stage preparatory for acclimation, and a late stage of new homeostasis with reduced growth.

Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene

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.

Identification of R-Gene Homologous DNA Fragments Genetically Linked to Disease Resistance Loci in Arabidopsis thaliana

Disease resistance in plants is a desirable economic trait. A number of disease resistance genes from various plant species have been cloned so far. The gene products of some of these can be distinguished by the presence of an N-terminal nucleotide binding site and a C-terminal stretch of leucine-rich repeats. Although these gene products are structurally related, the DNA sequences are poorly conserved. Only parts of the nucleotide binding site share enough DNA identity to design primers for polymerase chain reaction amplification of related DNA sequences. Such primers were used to amplify different resistance-gene-like (RGL) DNA fragments from Arabidopsis thaliana accessions Landsberg erecta and Columbia. Almost all cloned DNA fragments were genetically closely linked with known disease resistance loci. Most RGL fragments were found in a clustered or dispersed multi-copy sequence organization, supporting the supposed correlation of RGL sequences and disease resistance loci.

Rice mutant resources for gene discovery

With the completion of genomic sequencing of rice, rice has been firmly established as a model organism for both basic and applied research. The next challenge is to uncover the functions of genes predicted by sequence analysis. Considering the amount of effort and the diversity of disciplines required for functional analyses, extensive international collaboration is needed for this next goal. The aims of this review are to summarize the current status of rice mutant resources, key tools for functional analysis of genes, and our perspectives on how to accelerate rice gene discovery through collaboration.

Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1.

The a1 locus of Zea mays has been cloned using transposable elements as gene tags. The strategy was to make genomic libraries from maize stocks with a1 mutations induced either by En(Spm) or by Robertsons Mutator‐system. These libraries were then screened with either Spm‐I8 and En1, for the En‐containing mutant, or with Mu1 for the Mu‐induced mutation. There are many En and Mu1 hybridizing sequences present in the maize genome, however, by a process of cross‐screening of the positives from the two libraries and by molecular analysis of the En‐positive clones it was possible to identify clones in both libraries carrying all or part of the a1 gene.

ANTHOCYANINLESS2, a homeobox gene affecting anthocyanin distribution and root development in Arabidopsis.

The ANTHOCYANINLESS2 (ANL2) gene was isolated from Arabidopsis by using the maize Enhancer–Inhibitor transposon tagging system. Sequencing of the ANL2 gene showed that it encodes a homeodomain protein belonging to the HD–GLABRA2 group. As we report here, this homeobox gene is involved in the accumulation of anthocyanin and in root development. Histological observations of the anl2 mutant revealed that the accumulation of anthocyanin was greatly suppressed in subepidermal cells but only slightly reduced in epidermal cells. Furthermore, the primary roots of the anl2 mutant showed an aberrant cellular organization. We discuss a possible role of ANL2 in the accumulation of anthocyanin and cellular organization of the primary root.

Effects of Drought on Gene Expression in Maize Reproductive and Leaf Meristem Tissue Revealed by RNA-Seq

Drought stress affects cereals especially during the reproductive stage. The maize (Zea mays) drought transcriptome was studied using RNA-Seq analysis to compare drought-treated and well-watered fertilized ovary and basal leaf meristem tissue. More drought-responsive genes responded in the ovary compared with the leaf meristem. Gene Ontology enrichment analysis revealed a massive decrease in transcript abundance of cell division and cell cycle genes in the drought-stressed ovary only. Among Gene Ontology categories related to carbohydrate metabolism, changes in starch and Suc metabolism-related genes occurred in the ovary, consistent with a decrease in starch levels, and in Suc transporter function, with no comparable changes occurring in the leaf meristem. Abscisic acid (ABA)-related processes responded positively, but only in the ovaries. Related responses suggested the operation of low glucose sensing in drought-stressed ovaries. The data are discussed in the context of the susceptibility of maize kernel to drought stress leading to embryo abortion and the relative robustness of dividing vegetative tissue taken at the same time from the same plant subjected to the same conditions. Our working hypothesis involves signaling events associated with increased ABA levels, decreased glucose levels, disruption of ABA/sugar signaling, activation of programmed cell death/senescence through repression of a phospholipase C-mediated signaling pathway, and arrest of the cell cycle in the stressed ovary at 1 d after pollination. Increased invertase levels in the stressed leaf meristem, on the other hand, resulted in that tissue maintaining hexose levels at an “unstressed” level, and at lower ABA levels, which was correlated with successful resistance to drought stress.

Molecular analysis of the En/Spm transposable element system of Zea mays

The nucleotide sequence of the autonomous transposable element En‐1 isolated from the wx‐844::En‐1 allele has been determined. En‐1 is 8287 bp long. The structure of the mosaic gene 1, coding for the major En transcript, has been established. The promoter of gene 1 is located in the highly structured left end of the element and the gene spans almost the entire length of En‐1. The first intron of gene 1 is 4434 nucleotides long and contains two large open reading frames, 2714 bp and 761 bp in size, which hybridize to minor RNA species in Northern blot experiments.