Jeremy A. Roberts

The X-factor: visualizing undisturbed root architecture in soils using X-ray computed tomography

Although roots play a crucial role in plant growth and development through their acquisition and delivery of water and nutrients to the above-ground organs, our understanding of how they interact with their immediate soil environment largely remains a mystery as the opaque nature of soil has prevented undisturbed in situ root visualization (Perret et al., 2007). However, new developments in non-invasive techniques such as X-ray computed tomography (CT) provide, for the first time, an exciting opportunity to examine detailed root architecture in three dimensions (3-D) in undisturbed soil cores (Fig. 1). Although other non-invasive 3-D visualization procedures exist, X-ray CT is viewed as the most appropriate technique for studies of soil:root interactions as the presence of iron and manganese ions may provide interference when alternative techniques such as Nuclear Magnetic Resonance (NMR) are used (Heeraman et al., 1997). Detailed understanding of interactions between roots and their immediate soil environment is vital when considering issues such as land degradation as soil structure is a primary factor determining the availability of edaphic resources such as water and nutrients (Lynch, 1995), and is extrinsically linked to plant productivity (Moran et al., 2000). In view of the rapidly increasing human global population and the threat posed by climate change, maximizing crop yields and developing sustainable soil management strategies are vital for food security. X-ray CT overcomes some of the limitations associated with previous methodologies for studying roots by providing Fig. 1. X-ray CT image of roots of a 3-week-old Zea mays (L.) plant grown in a soil column (loamy sand, Newport series). 1 pixel1⁄444 lm.

Recent developments in abscission: shedding light on the shedding process

Plants are able to shed organs that are diseased, stressed or at the terminal stage of their development. Shedding takes place at a morphologically distinct site, the abscission zone, in a process that is accelerated by ethylene and retarded by auxin. Associated with cell separation is an increase in the expression of a spectrum of gene products, including hydrolytic enzymes and peptides that protect the exposed fracture surface from pathogenic attack. Key research objectives are the identification of abscission-specific gene promoters and the elucidation of the morphogenetic events that regulate cell differentiation in the abscission zone. Manipulation of both the site and rate of shedding will then become feasible.

Temporal and spatial expression of a polygalacturonase during leaf and flower abscission in oilseed rape and Arabidopsis.

During leaf abscission in oilseed rape (Brassica napus), cell wall degradation is brought about by the action of several hydrolytic enzymes. One of these is thought to be polygalacturonase (PG). Degenerate primers were used to isolate a PG cDNA fragment by reverse transcriptase-polymerase chain reaction from RNA extracted from ethylene-promoted leaf abscission zones (AZs), and in turn a full-length clone (CAW471) from an oilseed rape AZ cDNA library. The highest homology of this cDNA (82%) was to an Arabidopsis sequence that was predicted to encode a PG protein. Analysis of expression revealed that CAW471 mRNA accumulated in the AZ of leaves and reached a peak 24 h after ethylene treatment. Ethylene-promoted leaf abscission in oilseed rape was not apparent until 42 h after exposure to the gas, reaching 50% at 48 h and 100% by 56 h. In floral organ abscission, expression of CAW471 correlated with cell separation. Genomic libraries from oilseed rape and Arabidopsis were screened with CAW471 and the respective genomic clones PGAZBRAN and PGAZATisolated. Characterization of these PG genes revealed that they had substantial homology within both the coding regions and in the 5′-upstream sequences. Fusion of a 1,476-bp 5′-upstream sequence ofPGAZAT to β-glucuronidase or green fluorescent protein and transformation of Arabidopsis revealed that this fragment was sufficient to drive expression of these reporter genes in the AZs at the base of the anther filaments, petals, and sepals.

Increased 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Activity in Shoots of Flooded Tomato Plants Raises Ethylene Production to Physiologically Active Levels

Soil flooding increased 1-aminocyclopropane-1-carboxylic (ACC) acid oxidase activity in petioles of wild-type tomato (Lycopersicon esculentum L.) plants within 6 to 12 h in association with faster rates of ethylene production. Petioles of flooded plants transformed with an antisense construct to one isoform of an ACC oxidase gene (ACO1) produced less ethylene and had lower ACC oxidase activity than those of the wild type. Flooding promoted epinastic curvature but did so less strongly in plants transformed with the antisense construct than in the wild type. Exogenous ethylene, supplied to well-drained plants, also promoted epinastic curvature, but transformed and wild-type plants responded similarly. Flooding increased the specific delivery (flux) of ACC to the shoots (picomoles per second per square meter of leaf) in xylem sap flowing from the roots. The amounts were similar in both transformed and wild-type plants. These observations demonstrate that changes in ACC oxidase activity in shoot tissue resulting from either soil flooding or introducing ACC oxidase antisense constructs can influence rates of ethylene production to a physiologically significant extent. They also implicate systemic root to shoot signals in regulating the activity of ACC oxidase in the shoot.

A molecular and structural characterization of senescing Arabidopsis siliques and comparison of transcriptional profiles with senescing petals and leaves.

Senescence of plant organs is a genetically controlled process that regulates cell death to facilitate nutrient recovery and recycling, and frequently precedes, or is concomitant with, ripening of reproductive structures. In Arabidopsis thaliana, the seeds are contained within a silique, which is itself a photosynthetic organ in the early stages of development and undergoes a programme of senescence prior to dehiscence. A transcriptional analysis of the silique wall was undertaken to identify changes in gene expression during senescence and to correlate these events with ultrastructural changes. The study revealed that the most highly up-regulated genes in senescing silique wall tissues encoded seed storage proteins, and the significance of this finding is discussed. Global transcription profiles of senescing siliques were compared with those from senescing Arabidopsis leaf or petal tissues using microarray datasets and metabolic pathway analysis software (MapMan). In all three tissues, members of NAC and WRKY transcription factor families were up-regulated, but components of the shikimate and cell-wall biosynthetic pathways were down-regulated during senescence. Expression of genes encoding ethylene biosynthesis and action showed more similarity between senescing siliques and petals than between senescing siliques and leaves. Genes involved in autophagy were highly expressed in the late stages of death of all plant tissues studied, but not always during the preceding remobilization phase of senescence. Analyses showed that, during senescence, silique wall tissues exhibited more transcriptional features in common with petals than with leaves. The shared and distinct regulatory events associated with senescence in the three organs are evaluated and discussed.

Flower abscission in mutant tomato plants

The effect of two mutations of the tomato known as Never ripe (Nr) and ripening inhibitor (rin) on abscission of the flowers was investigated. In the presence of ethylene the rate of abscission of normal and rin explants was similar, while that of Nr explants was delayed. The appearance and subsequent increases in both polygalacturonase (EC 3.2.1.15) and β-1-4-glucanase (EC 3.2.1.4) enzyme activities were similar in normal and rin explants, but retarded in Nr explants. Of these two, only polygalacturonase activity was exclusively associated with abscission-zone tissue.

Characterisation of an mRNA encoding a metallothionein-like protein that accumulates during ethylene-promoted abscission of Sambucus nigra L. leaflets

A cDNA encoding a metallothionein-like protein has been isolated from a cDNA library from the abscission zones of ethylene-treated Sambucus nigra leaflets. The precise function of this group of proteins in plants has yet to be confirmed but in animals there is convincing evidence that they bind heavy metals. Several of these proteins have recently been characterised from plants and it has been demonstrated that heavy metals have no stimulatory effect on their expression. In this paper we describe the isolation and characterisation of a metallothionein-like mRNA identified as a consequence of differentially screening a cDNA library for messages up-regulated during abscission. The accumulation of the mRNA occurred in the abscission zone tissue within 18 h of exposure to ethylene while, in contrast, no expression was detectable in adjacent non-abscission-zone tissue. The transcript size of the message was approximately 0.6 kb. Northern analysis revealed that the cDNA insert (JET12) did not hybridise to mRNA from either green or senescing leaflets but a signal was detectable with mRNA extracted from senescent tissue. The size of this hybridising transcript was approximately 0.5 kb. The predicted metallothionein-like protein encoded by JET12 was cysteinerich (18.4%) and had a molecular weight of approximately 7.5 kDa. Southern analysis of S. nigra genomic DNA showed that the mRNA was encoded by a small gene family. The protein exhibited greatest homology to other metallothioneins belonging to the Type 2 family including those from Mimulus (62%) and Arabidopsis (57%). This homology was greatest around the cysteine-rich amino and carboxy termini. The possible role of the protein encoded by JET12 during ethylene-promoted leaflet abscission is discussed.

Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants.

Polygalacturonase (PG, EC 3.2.1.15), an enzyme commonly found in ripening fruit, has also been shown to be associated with abscission. A zone-specific rise in PG activity accompanies the abscission of both leaves and flowers of tomato (Lycopersicon esculentum Mill.) plants. Studies of transgenic plants expressing an antisense RNA for fruit PG indicate that although the enzyme activity in transgenic fruit is < 1 % of that in untransformed fruit, the PG activity in the leaf abscission zone increases during separation to a similar value to that in untransformed plants. The timing and rate of leaf abscission in transgenic plants are unaffected by the introduction of the antisense gene. A polyclonal antibody raised against tomato fruit PG does not recognise the leaf abscission protein. Furthermore a complementary DNA (cDNA) clone (pTOM6), which has been demonstrated to code for fruit PG, does not hybridise to mRNA isolated from the abscission-zone region of tomato leaves. These results indicate that the PG protein in abscission zones of tomato is different from that in the fruit, and that the gene coding for this protein may also be different.

The potential for underutilized crops to improve security of food production

Staple crops face major challenges in the near future and a diversification away from over-reliance on staples will be important as part of the progress towards the goal of achieving security of food production. Underutilized or neglected crops species are often indigenous ancient crop species which are still used at some level within the local, national or even international communities, but have the potential to contribute further to the mix of food sources than they currently do. The most cost-effective and easily disseminated changes that can be made to a crop are changes to the genetics, as these are contained within the seed itself and, for many species, the seed is a pure breeding, self-replicating, resource. This article focuses on the potential of underutilized crops to contribute to food security and, in particular, whether genetics and breeding can overcome some of the constraints to the enhanced uptake of these species in the future. The focus here is on overview rather than detail and subsequent articles will examine the current evidence base.

Ethylene-promoted tomato flower abscission and the possible involvement of an inhibitor

The abscission zone in tomato (Lycopersicon esculentum (L.) Mill. flower pedicels is morphologically distinguishable prior to separation and is delineated by an indentation of the epidermis. Exposure of excised pedicels with the flower attached to ethylene results in abscission within 12 h and this can be accelerated by flower removal. Abscission of excised pedicels with the flower removed takes place in the absence of exogenous ethylene but this is delayed by pretreatment with aminoethoxyvinyl glycine, an inhibitor of ethylene biosynthesis. The data presented support the hypothesis that flower tissue is the source of an abscission inhibitor.