Lindy A. Brigham

Differential Expression of Proteins and mRNAs from Border Cells and Root Tips of Pea.

Many plants release large numbers of metabolically active root border cells into the rhizosphere. We have proposed that border cells, cells produced by the root cap meristem that separate from the rest of the root upon reaching the periphery of the cap, are a singularly differentiated part of the root system that modulates the environment of the plant root by producing specific substances to be released into the rhizosphere. Proteins synthesized in border cells exhibit profiles that are very distinct from those of the root tip (root cap, root meristem, and adjacent cells). In vivo-labeling experiments demonstrate that 13% of the proteins that are abundant in preparations from border cells are undetectable in root tip preparations. Twenty-five percent of the proteins synthesized by border cells in a 1-h period are rapidly excreted into the incubation medium. Quantitative variation in levels of specific marker proteins, including glutamine synthetase, heat-shock protein 70, and isoflavone reductase, also occurs between border cells and cells in the root tip. mRNA differential-display assays demonstrate that these large qualitative and quantitative differences in protein expression are correlated with similarly distinct patterns of gene expression. These observations are consistent with the hypothesis that a major switch in gene expression accompanies differentiation into root border cells, as expected for cells with specialized functions in plant development.

Root border cells as tools in plant cell studies.

Publisher Summary This chapter focuses on the use of root border cells as tools in plant cell studies. Root border cells lend themselves to a wide range of studies by virtue of their unique morphology. Border cells are populations of single cells that separate from the outer cells of the root cap and can be collected by gentle agitation in water. Substantial evidence is consistent with the hypothesis that border cells constitute a uniquely differentiated part of the root system that functions to modulate microbial populations in the rhizosphere. However, as populations of single, metabolically active cells with intact cell walls, root border cells provide tools to study a wide range of biological questions. Unlike protoplasts, border cells survive in a wide range of osmotic conditions, including distilled water, without lysis or observable cellular damage. The chapter provides specific conditions for the manipulation of pea root border cells. Diverse applications of root border cells in assays for research and teaching are also discussed.

PRIMARY STRUCTURE OF THE MRNA ENCODING A 16.5-KDA UBIQUITIN-CONJUGATING ENZYME OF PISUM SATIVUM

The complementary DNA (PsU BC4) representing an mRNA encoding an ubiquitin-conjugating enzyme (UBC) of Pisum sativum has been cloned. The coding region is 444 nucleotides (nt) in length and capable of specifying a 16.5-kDa protein of 148 amino acids (aa) with an isoelectric point of 7.95. The deduced aa sequence showed 97% identity with Arabidopsis thaliana AtUBC8-12 families and tomato ERT17, and 80% identity with yeast ScUBC4 and ScUBC5 and Drosophila melanogaster DmUBC4. The active site cysteine (Cys85) found in UBCs so far described is also conserved in the P. sativum sequence.

Expression of transferred genes during hairy root development in pea

Root border cell development and expression of reporter genes were evaluated in transgenic pea hairy roots. Successful induction of hairy roots in pea is conditioned by bacterial strain and plant genotype, as well as by developmental and environmental factors. Morphological changes sometimes occur when hairy roots are transferred from infected plants to tissue culture media, but such changes are confined to specific clones. Expression of reporter genes under the control of promoters from bean (Phaseolus vulgaris L.) “stress” genes encoding phenylalanine ammonia lyase and chalcone synthase were evaluated. Expression patterns vary between hairy roots taken directly from infected plants, and those grown in culture; most hairy roots taken from infected plants exhibit expression throughout all tissues, whereas expression in cultured hairy roots is most often localized to specific tissues. Patterns of expression that occur during different stages of hairy root development are very similar to those observed in transgenic plants expressing the same fusion genes. Border cell separation and release in hairy roots is normal, and expression of glucuronidase in border cells of some transgenic roots resulted in development of bright blue single cells. Cultured hairy roots should provide a very useful model for studying the effect of defined changes in root border cells on microbial associations with roots of this important legume.

Molecular cloning and expression of mRNAs encoding H1 histone and an H1 histone-like sequences in root tips of pea (Pisum sativum L.)

Two cDNA clones representing mRNAs, highly expressed in pea root tips, were isolated by mRNA differential display. Ribonuclease protection analyses showed different patterns of expression of these two messages in several pea tissues. Sequence analysis showed that the first clone, PsH1b-40, has 100% homology with a previously isolated H1 histone cDNA, PsH1b. However, it has an additional 30 nt at the 3′ end which is absent in PsH1b, suggesting possible multiple polyadenylation sites in the same mRNA. The second clone, PsH1b-41, encodes a deduced 19.5 kDa protein of 185 amino acids with an isoelectric point of 11.5. The putative globular domain of the encoded protein showed 67–71% residue identity with globular domains of 28 kDa pea PsH1b H1 histone and Arabidopsis thaliana H1-1 H1 histone. It has 9 repeating motifs of (T/S)XXK. In the C-terminal domain, there are four lysine-rich repeating motifs of SXK(T/S)PXKKXK which may be involved in chromatin condensation and decondensation. Southern blot analysis of nuclear DNA shows that PsH1-41 belongs to a multigene family.

In-cell RT-PCR in a single, detached plant cell

A procedure is presented for the detection of mRNA in a single root border cell of pea seedlings using RT-PCR (reverse transcription-polymerase chain reaction) and direct incorporation of digoxigenin-dUTP. It includes cell preservation with aldehyde. Signal is detected using a conjugate of anti-DIG and alkaline phosphatase.

Altered growth and root tip morphology in Pisum sativum L. in response to altered expression of a gene expressed in border cells

Background and aimsRecent studies suggest that root border cells function in defense of the root by an extracellular DNA-based trapping mechanism similar to that described in mammalian white blood cells. Genes controlling the specialized properties of border cells as they detach from the root tip therefore are of interest.MethodsmRNA differential display was used to identify Brd13, a sequence expressed in border cells but not other root tissues. RNase protection and mRNA Northern blot analyses, and reporter gene expression under the control of the Brd13 promoter in transgenic hairy roots were used to confirm localized expression. Phenotype analysis of transgenic hairy roots expressing Brd13 antisense mRNA was carried out.ResultsBrd13 was expressed constitutively in border cells but not in leaves, stems, or roots without border cells. The predicted protein shares sequence similarity with flavin-binding proteins. Transgenic hairy roots expressing Brd13 antisense mRNA exhibited abnormal growth and morphology.ConclusionsWe report here that altered expression of a putative flavin-binding protein in border cells resulted in altered root development. Flavin-binding proteins play key roles in development, defense, and local auxin biosynthesis. The Brd13 gene and its promoter may be useful in creating defined changes in root development and defense.