Stomatal pores on the leaf surface control both the uptake of CO2 for photosynthesis and the loss of water during transpiration. Since the industrial revolution, decreases in stomatal numbers in parallel with increases in atmospheric CO2 concentration have provided evidence of plant responses to changes in CO2 levels caused by human activity. This inverse correlation between stomatal density and CO2 concentration also holds for fossil material from the past 400 million years and has provided clues to the causes of global extinction events. Here we report the identification of the Arabidopsis gene HIC (for high carbon dioxide), which encodes a negative regulator of stomatal development that responds to CO2 concentration. This gene encodes a putative 3-keto acyl coenzyme A synthase--an enzyme involved in the synthesis of very-long-chain fatty acids. Mutant hic plants exhibit up to a 42% increase in stomatal density in response to a doubling of CO2. Our results identify a gene involved in the signal transduction pathway responsible for controlling stomatal numbers at elevated CO2.

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Sedentary plant-parasitic nematodes are able to induce specialized feeding structures in the root system of their host plants by triggering a series of dramatic cellular responses. These changes presumably are accompanied by a reprogramming of gene expression. To monitor such changes, a variety of promoter-gusA fusion constructs were introduced into Arabidopsis and tobacco. Transgenic plants were analysed histochemically for GUS activity in the nematode feeding structures after infection with either Heterodera schachtii or Meloidogyne incognita. Promoters of the Cauliflower Mosaic Virus 35S gene, the bacterial nopaline synthase, rooting loci (rol) and T-cyt genes and the plant-derived phenylalanine ammonia-lyase I gene, which are highly active in non-infected roots, were all downregulated in the feeding structures as indicated by the strong decrease of GUS activity inside these structures. Less stringent downregulation was observed with chimeric gusA fusion constructs harbouring truncated rolB and rolC promoter sequences. Similar observations were made with transgenic Arabidopsis lines that carried randomly integrated promoterless gusA constructs to identify regulatory sequences in the plant genome. Most of the lines that were selected for expression in the root vascular cylinder demonstrated local downregulation in feeding structures after infection with H. schachtii. The reverse pattern of GUS activity, a blue feeding structure amidst unstained root cells, was also found in several lines. However, GUS activity that was entirely specific for the feeding structures was not observed. Our data show that the expression of a large number of genes is influenced during the development of the nematode feeding structures.

Summary We have established culture conditions for successful infection and development of several economically important cyst‐forming and root‐knot nematodes on Arabidopsis thaliana under monoxenic conditions. Complete life cycles were obtained with the sedentary cyst nematodes Heterodera schachtii, H. trifolii, H. cajani and the root‐knot nematodes Meloidogyne incognita and M. arenariaas well as with the migratory nematode Pratylenchus penetrans. In contrast, H. goettingiana and Globodera rostochiensis were unable to develop on Arabidopsis roots. Tissue‐culture quality agar and medium conditions optimized for hydroponic root culture were essential for successful infections. Detailed in‐vivo observations were made inside Arabidopsis roots during the early infection stages of M. incognita and during complete development of H. schachtii. Seventy‐four different ecotypes of Arabidopsis were screened for their susceptibility towards H. schachtii resulting in a range of infection rates. None of the ecotypes tested showed complete resistance in vitro. The use of Arabidopsis as a host for plant‐parasitic nematodes will provide a new model system for the molecular genetic analysis of this interaction.

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