On systems thinking, systems biology and the in Silico Plant

The recent summary report of a Department of Energy Workshop on Plant Systems Biology (P.V. Minorsky [2003] Plant Physiol 132: 404-409) offered a welcomed advocacy for systems analysis as essential in understanding plant development, growth, and production. The goal of the Workshop was to consider methods for relating the results of molecular research to real-world challenges in plant production for increased food supplies, alternative energy sources, and environmental improvement. The rather surprising feature of this report, however, was that the Workshop largely overlooked the rich history of plant systems analysis extending over nearly 40 years (Sinclair and Seligman, 1996) that has considered exactly those challenges targeted by the Workshop. Past systems research has explored and incorporated biochemical and physiological knowledge into plant simulation models from a number of perspectives. The research has resulted in considerable understanding and insight about how to simulate plant systems and the relative contribution of various factors in influencing plant production. These past activities have contributed directly to research focused on solving the problems of increasing biomass production and crop yields. These modeling approaches are also now providing an avenue to enhance integration of molecular genetic technologies in plant improvement (Hammer et al., 2002).

Using the canonical modelling approach to simplify the simulation of function in functional-structural plant models

Functional-structural plant models that include detailed mechanistic representation of underlying physiological processes can be expensive to construct and the resulting models can also be extremely complicated. On the other hand, purely empirical models are not able to simulate plant adaptability and response to different conditions. In this paper, we present an intermediate approach to modelling plant function that can simulate plant response without requiring detailed knowledge of underlying physiology. Plant function is modelled using a 'canonical' modelling approach, which uses compartment models with flux functions of a standard mathematical form, while plant structure is modelled using L-systems. Two modelling examples are used to demonstrate that canonical modelling can be used in conjunction with L-systems to create functional-structural plant models where function is represented either in an accurate and descriptive way, or in a more mechanistic and explanatory way. We conclude that canonical modelling provides a useful, flexible and relatively simple approach to modelling plant function at an intermediate level of abstraction.

Functional-structural plant modelling using a combination of architectural analysis, L-systems and a canonical model of function

This paper presents a new method for producing a functional-structural plant model that simulates response to different growth conditions, yet does not require detailed knowledge of underlying physiology. The example used to present this method is the modelling of the mountain birch tree. This new functional-structural modelling approach is based on linking an L-system representation of the dynamic structure of the plant with a canonical mathematical model of plant function. Growth indicated by the canonical model is allocated to the structural model according to probabilistic growth rules, such as rules for the placement and length of new shoots, which were derived from an analysis of architectural data. The main advantage of the approach is that it is relatively simple compared to the prevalent process-based functional-structural plant models and does not require a detailed understanding of underlying physiological processes, yet it is able to capture important aspects of plant function and adaptability, unlike simple empirical models. This approach, combining canonical modelling, architectural analysis and L-systems, thus fills the important role of providing an intermediate level of abstraction between the two extremes of deeply mechanistic process-based modelling and purely empirical modelling. We also investigated the relative importance of various aspects of this integrated modelling approach by analysing the sensitivity of the standard birch model to a number of variations in its parameters, functions and algorithms. The results show that using light as the sole factor determining the structural location of new growth gives satisfactory results. Including the influence of additional regulating factors made little difference to global characteristics of the emergent architecture. Changing the form of the probability functions and using alternative methods for choosing the sites of new growth also had little effect. (c) 2004 Elsevier B.V. All rights reserved.

The SEN1 gene of Arabidopsis is regulated by signals that link plant defence responses and senescence

Plant defence and senescence share many similarities as evidenced by extensive co-regulation of many genes during these responses. To better understand the nature of signals that are common to plant defence and senescence, we studied the regulation of SEN1 encoding a senescence-associated protein during plant defence responses in Arabidopsis. Pathogen inoculations and treatments with defence-related chemical signals, salicylic acid and methyl jasmonate induced changes in SEN1 transcript levels. Analysis of transgenic plants expressing the SEN1 promoter fused to uidA reporter gene confirmed the responsiveness of the SEN1 promoter to defence- and senescence-associated signals. Expression analysis of SEN1 in a number of defence signalling mutants indicated that activation of this gene by pathogen occurs predominantly via the salicylic and jasmonic acid signalling pathways, involving the functions of EDS5, NPR1 and JAR1 In addition, in the absence of pathogen challenge, the cpr5/hys1 mutant showed elevated SEN1 expression and displayed an accelerated senescence response following inoculation with the necrotrophic fungal pathogen Fusarhan oxysporum. Although the analysis of the sen1-1 knock-out mutant did not reveal any obvious role for this gene in defence or senescence-associated events, our results presented here show that SEN1 is regulated by signals that link plant defence and senescence responses and thus represents a useful marker gene to study the overlap between these two important physiological events. (c) 2005 Elsevier SAS. All rights reserved.

Development of PCR-based markers for detection of Leifsonia xyli subsp. xyli in fibrovascular fluid of infected sugarcane plants

DNA of Leifsonia xyli subsp. xyli (Lxx), the causal agent of ratoon stunting disease of sugarcane, was detected in the fibrovascular fluid of sugarcane plants using random amplified polymorphic DNA PCR-based amplification using two 10-mer oligonucleotide primers. The primers OPC-02 and OPC-11 produced Lxx-specific markers of approximately 800 bp and 1000 bp, respectively. A cloned DNA fragment from the 800 bp PCR product (pSKC2-800) hybridised to a single genomic DNA fragment from Lxx when used as a probe in Southern hybridisation. This cloned fragment did not hybridise to L. xyli subsp. cynodontis (Lxc), or L. xyli-like bacteria isolated from grasses in Australia, indicating the usefulness of this DNA fragment as a specific probe for Lxx. A cloned fragment from the 1000 bp PCR product ( pSKC11-1000) hybridised to three genomic fragments in Lxx isolates, one genomic fragment in two of the four isolates of L. xyli-like bacteria, and in two of the four isolates of Lxc isolated from the USA. These results indicate that L. xyli-like bacteria are more likely to be related to Lxc than Lxx. These probes did not hybridise to the DNA from strains of the species of Clavibacter, Rathayibacter, Acidovorax, Ralstonia, Pseudomonas and Xanthomonas tested. Two oligonucleotide primers (21-mer) designed from the pSKC2-800 sequences specifically amplified template DNA from Lxx and detected as few as 5 x 10(4) cells/mL in fibrovascular fluid from sugarcane plants infected with Lxx.

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30%, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K oxytoca ATCC 13182(T) to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His(78), HiS(125), HiS(141) and His(189)) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95%. DEPC treatment increased absorbance at 240-244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that HiS125 might be the residue required for albicidin binding. Mutation of HiS125 to either alanine or leucine resulted in about 32% loss of binding activity, and deletion of HiS125 totally abolished binding activity. Mutation of HiS125 to arginine and tyrosine had no effect. These results indicate that HiS125 plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.

Utility of cotyledon and detached leaf assays for assessing root reactions of lucerne to Phytophthora root rot caused by Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne production but it can be managed through the use of resistant cultivars. Current resistance screening methods, using mature plants or post-emergence seedling assays, are costly and time consuming. The use of zoospore inoculum on detached leaves and intact cotyledons as an assay for plant resistance was assessed using genetically defined segregating populations. The detached leaf assay was a reproducible test, but this test could not be used for accurately predicting root ratings. The cotyledon tests using zoospores gave results at the population level that were indicative of the root responses of 19 cultivars and lines tested. The cotyledon reaction of individual plants also showed a strong association with root response. The cotyledon test, while not completely predictive of mature root responses, allowed the selection of Phytophthora resistant plants at a higher frequency than could be achieved by random selection.

Characterisation of three ACC synthase gene family members during post-harvest-induced senescence in broccoli (Brassica oleracea L. var. italica)

We investigated the gene expression profiles of different members of the 1-aminocyclopropane-1-carboxilic acid (ACC) synthase (EC 4.4.1.14) gene family in broccoli (Brassica oleracea L. var. italica) during the post-harvest-induced senescence process. Using RT-PCR, three different cDNAs coding for ACC synthase (BROCACS1, BROCACS2 and BROCACS3) were amplified from floret tissue at the start of the senescence process. The three genes share relatively little homology, but have highly homologous sequences in Arabidopsis thaliana, and could be functionally related to these counterparts. Southern analyses suggest that BROCACS1 and BROCACS3 are present as single copy genes, while there are probably two copies of BROCACS2. All three genes showed different expression patterns: BROCACS1 is likely to be either wound - or mechanical stress-induced showing high transcript levels after harvesting, but no detectable expression afterwards. BROCACS2 shows steady expression throughout senescence, increasing at the latest stages, and BROCACS3 is almost undetectable until the final stages. Our results suggest that BROCACS1 could be required to initiate the senescence process, while BROCACS2 would be the main ACC synthase gene involved throughout the post-harvest-induced senescence. BROCACS3's expression pattern indicates that it is not directly involved in the initial stages of senescence, but in the final remobilization of cellular resources.