David F. Hildebrand

Evidence supporting a role of jasmonic acid in Arabidopsis leaf senescence

In this work, the role of jasmonic acid (JA) in leaf senescence is examined. Exogenous application of JA caused premature senescence in attached and detached leaves in wild-type Arabidopsis but failed to induce precocious senescence of JA-insensitive mutantcoi1 plants, suggesting that the JA-signaling pathway is required for JA to promote leaf senescence. JA levels in senescing leaves are 4-fold higher than in non-senescing ones. Concurrent with the increase in JA level in senescing leaves, genes encoding the enzymes that catalyze most of the reactions of the JA biosynthetic pathway are differentially activated during leaf senescence in Arabidopsis, except for allene oxide synthase, which is constitutively and highly expressed throughout leaf development. Arabidopsis lipoxygenase 1 (cytoplasmic) expression is greatly increased but lipoxygenase 2 (plastidial) expression is sharply reduced during leaf senescence. Similarly,AOC1 (allene oxide cyclase 1),AOC2, and AOC3 are all up-regulated, whereas AOC4 is down-regulated with the progression of leaf senescence. The transcript levels of 12-oxo-PDA reductase 1 and 12-oxo-PDA reductase 3 also increase in senescing leaves, as does PED1 (encoding a 3-keto-acyl-thiolase for β-oxidation). This represents the first report, to our knowledge, of an increase in JA levels and expression of oxylipin genes during leaf senescence, and indicates that JA may play a role in the senescence program.

Design and construction of a versatile system for the expression of foreign genes in plants.

We have built a series of vectors to allow the constitutive or light-regulated expression of foreign genes in plants. These vectors carry expression cassettes consisting of either the cauliflower mosaic virus 35S promoter or the pea rbcS-E9 promoter, a multiple cloning site derived from M13um20, and the rbcS-E9 polyadenylation site. These cassettes have been incorporated into pBR322-based or RK2-based replicons to facilitate direct DNA uptake or Agrobacterium tumefaciens-mediated gene transfer. Their application for the expression of a bacterial gene is described.

Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in arabidopsis.

Multiple forms of phospholipase D (PLD) were activated in response to wounding, and the expressions of PLDα, PLDβ, and PLDγ differed in wounded Arabidopsis leaves. Antisense abrogation of the common plant PLD, PLDα, decreased the wound induction of phosphatidic acid, jasmonic acid (JA), and a JA-regulated gene for vegetative storage protein. Examination of the genes involved in the initial steps of oxylipin synthesis revealed that abrogation of the PLDα attenuated the wound-induced expression of lipoxygenase 2 (LOX2) but had no effect on allene oxide synthase (AOS) or hydroperoxide lyase in wounded leaves. The systemic induction of LOX2, AOS, and vegetative storage protein was lower in the PLDα-suppressed plants than in wild-type plants, with AOS exhibiting a distinct pattern. These results indicate that activation of PLD mediates wound induction of JA and that LOX2 is probably a downstream target through which PLD promotes the production of JA.

Activation of a Stress-Responsive Mitogen-Activated Protein Kinase Cascade Induces the Biosynthesis of Ethylene in Plants

Plants under stress from both biotic and abiotic sources produce increased levels of ethylene, which is perceived by ethylene receptors and triggers cellular responses further downstream. Protein phosphorylation and dephosphorylation were implicated in the regulation of ethylene induction by stresses based on studies using protein kinase and phosphatase inhibitors. However, the kinase(s) involved remains to be determined. Using a conditional gain-of-function transgenic system, we demonstrate that the activation of SIPK, a tobacco mitogen-activated protein kinase (MAPK), by NtMEK2DD, an active mutant of the upstream kinase of SIPK, resulted in a dramatic increase in ethylene production. The increase in ethylene after the activation of SIPK coincided with a dramatic increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity, which was followed by the activation of a subgroup of ACS and ACC oxidase (ACO) genes, suggesting that either the activation of unidentified ACS(s) or post-transcriptional regulation is involved. Infection with Tobacco mosaic virus (TMV), which is known to activate the SIPK cascade and induce ethylene biosynthesis, also induced the same ACSs and ACOs. After ethylene production in NtMEK2DD plants, strong activation of ETHYLENE-RESPONSE FACTOR (ERF) genes was observed, similar to the effect in NN tobacco plants infected with TMV. In contrast to previous reports, no major increase in jasmonic acid (JA) and methyl jasmonate (MJ) was detected after the activation of SIPK/WIPK in NtMEK2DD transgenic plants. These results suggest that the induction of ethylene but not JA/MJ is involved in plant defense responses mediated by the NtMEK2-SIPK/WIPK pathway.

The soybean 94-kilodalton vegetative storage protein is a lipoxygenase that is localized in paraveinal mesophyll cell vacuoles.

Soybean leaves contain three proteins (the vegetative storage proteins or VSPs) that respond to nitrogen status and are believed to be involved in the temporary storage of nitrogen. One of these proteins, with a molecular mass of 94 kD and termed vsp94, was microsequenced. Partial amino acid sequence indicated that vsp94 was highly homologous to the lipoxygenase protein family. Further evidence that vsp94 is a lipoxygenase was obtained by demonstrating that vsp94 cross-reacted with a lipoxygenase antibody. Also, a lipoxygenase cDNA coding region was able to detect changes in an mRNA that closely parallel changes in vsp94 protein levels resulting from alteration of nitrogen sinks. Extensive immunocytochemical data indicate that this vsp94/lipoxygenase is primarily expressed in the paraveinal mesophyll cells and is subcellularly localized in the vacuole. These observations are significant in that they suggest that plant lipoxygenases may be bifunctional proteins able to function enzymatically in the hydroperoxidation of lipids and also to serve a role in the temporary storage of nitrogen during vegetative growth.

The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis.

In plants, changes in the levels of oleic acid (18:1), a major monounsaturated fatty acid (FA), results in the alteration of salicylic acid (SA)- and jasmonic acid (JA)-mediated defense responses. This is evident in the Arabidopsisssi2/fab2 mutant, which encodes a defective stearoyl-acyl carrier protein-desaturase (S-ACP-DES) and consequently accumulates high levels of stearic acid (18:0) and low levels of 18:1. In addition to SSI2, the Arabidopsis genome encodes six S-ACP-DES-like enzymes, the native expression levels of which are unable to compensate for a loss-of-function mutation in ssi2. The presence of low levels of 18:1 in the fab2 null mutant indicates that one or more S-ACP-DES isozymes contribute to the 18:1 pool. Biochemical assays show that in addition to SSI2, four other isozymes are capable of desaturating 18:0-ACP but with greatly reduced specific activities, which likely explains the inability of these SSI2 isozymes to substitute for a defective ssi2. Lines containing T-DNA insertions in S-ACP-DES1 and S-ACP-DES4 show that they are altered in their lipid profile but contain normal 18:1 levels. However, overexpression of the S-ACP-DES1 isoform in ssi2 plants results in restoration of 18:1 levels and thereby rescues all ssi2-associated phenotypes. Thus, high expression of a low specific activity S-ACP-DES is required to compensate for a mutation in ssi2. Transcript level of S-ACP-DES isoforms is reduced in high 18:1-containing plants. Enzyme activities of the desaturase isoforms in a 5-fold excess of 18:1-ACP show product inhibition of up to 73%. Together these data indicate that 18:1 levels are regulated at both transcriptional and post-translational levels.

A procedure for plant regeneration from immature cotyledon tissue of soybean

For some time, soybean (Glycine max (L.) Merr.) has been conspicuous among major crops in lacking an in vitro regeneration system. This deficiency has precluded the application of many nonconventional techniques of crop improvement (See Abelson, 1983; Lea and Stewart, 1984; Hildebrand et al., 1985 for reviews). Although soybean is recalcitrant in vitro, some wild Glycine species are more responsive. Of these, Glycine canescens F. J. Herm appears most amenable; shoot production has been obtained from hypocotyl (Kameya and Widholm, 1981; Widholm and Rick, 1983), cotyledon (Widholm and Rick, 1983; Grant, 1984), leaf and flower tissues (Lazzeri, Hildebrand and Collins, unpublished) and also from protoplasts (Newell and Luu, 1985; Myers, Lazzeri and Collins, unpublished). Shoot regeneration has also been reported in two other wild species, G. tomentella Mayata (Kameya and Widholm, 1981) and G.clandestina Wendl. (Hymowitz et al., 1986).

Recovery of primary transformants of soybean

Three transformants of soybean, Glycine max (L.) Merr., have been recovered among a total of 18 plants regenerated by somatic embryogenesis from immature cotyledon tissues after cocultivation with Agrobacterium strains carrying a 15 kD zein gene (pH5PZ3D). DNA from upper leaves hybridized to a synthetic RNA probe specific for the zein sequence at a level equivalent to at least one copy per haploid genome. Hybridization to a vir G/C probe, however, was negligible, indicating that sequestration of whole bacteria or even persistence of plasmids within the tissues could not account for the zein hybridization signals. Progeny of all plants were uniformly untransformed. Since most somatic embryos have a multicellular origin in the regeneration system used, it is believed that the primary transformants were chimeric. The results indicate that somatic embryogenesis may be adaptable to Agrobacterium-mediated transformation in soybean, but that greater numbers of mitotic cycles under selection before embryo initiation will be required if somatic embryogenesis is to be used efficiently for production of plants with transformed germ-line cells.

Soybean somatic embryogenesis: Effects of hormones and culture manipulations

Somatic embryos were induced in cultures of immature soybean (Glycine max (L.) Merr) embryos, or isolated cotyledons on MS modified medium supplemented with NAA and 2,4-D, BAP and ABA. When NAA and 2,4-D were compared at similar concentrations (25 and 23 μM), 2,4-D produced larger number of somatic embryos, however, embryogenesis efficiency was improved in media containing NAA by using higher levels (100–150 μM) of the auxin. Somatic embryo morphology varied with auxin type: NAA-induced embryos more closely resembled zygotic embryos than did 2,4-D-induced embryos. Additions of BAP or ABA to auxin-containing media had either no effect or reduced embryo production, although ABA altered the morphology of 2,4-D-induced embryos. In media containing both NAA and 2,4-D, the latter was dominant in terms of embryo morphology. The effects of subculture frequency and of transfers between 2,4-D and NAA media were investigated: Subculture frequency influenced mainly the frequency of normal embryos, while preculture on 2,4-D increased subsequent embryogenesis efficiency on NAA medium but reduced the frequency of normal embryos.

Fatty acid alteration by a 9 desaturase in transgenic plant tissue

Nicotiana tabacum tissue was transformed with a rat stearyl–CoA desaturase gene. Gas Chromatographie analysis showed an increase in monounsaturated 16 and 18 carbon fatty acids in selected transformed calli and leaves. Fractionation of lipid classes indicated that palmitoleic acid was found in the phosphatidylcholine fraction of desaturase–transformed leaves, but not in leaves transformed with vector sequences. Plant transformation was verified by polymerase chain reaction (PCR) amplification of total leaf DNA.