JA and ethylene pathways related to PCD. Little is known concerning the role of JA in either seed development or PCD. Subcluster 1(2) genes that are upregulated in the degenerating maternal tissue include transcripts involved in both JA and ethylene biosynthesis. Their gene products are assumed to increase levels of jasmonate and ethylene in the pathways depicted in Figure 3(b). JA biosynthesis begins with a GDSL-motif lipase (CA020570, BQ469565, CA024107) and other lipase gene family members (CA020586, AL511679, CA020727, BU980997), together with lipid transfer proteins [CA025922, CA022749, CA021042, CA027579, CA022845, BU982975, see Table S2, cluster 1(2)], which are abundantly expressed during late pericarp development. Their precise functions remain unknown. The co-expression of lipoxygenase 2 genes (CA027764, CA024178, CA020483, CA022863, CA025433, BU974281) obviously reflects their participation in the supply of 13-hydroxy-9,11,15-octadecatrienoic acid (13-HPOT), which is involved in PCD (Bachmann et al., 2002; Maccarrone et al., 2001). Taken together, these observations suggest that lipolysis and the JA biosynthetic pathway are initiated in the senescing pericarp tissue. Also co-expressed are allene oxide synthase 1 and 2 genes (CA021313, AL511064), which have been shown to require 13-HPOT as substrate (Maucher et al., 2000). As a result of the activity of allene oxide cyclase (AOC), 12-oxo-phytodienoic acid (OPDA) is formed from highly unstable epoxide. The increase in AOC expression during late pericarp development correlates with increased OPDA levels, as confirmed by metabolite measurements (Figure 3b). CA021001 shares significant homology with tomato/Arabidopsis OPR3 (OPDA reductase 3) and is up-regulated in the pericarp. The OPR3 gene product specifically recognizes cis-(+)-OPDA (Schaller et al., 2000), which is transformed into JA by sequential β-oxidation. Thus the elevated levels of JA in the maturing pericarp (Figure 3b), also observed in soybean seeds (Simpson and Gardner, 1995), emphasize its importance in mediating PCD in pericarp tissue. JA has also been shown to promote leaf senescence (He et al., 2002), a comparable process. Although little is known concerning the key regulators of JA signaling networks in seeds, the TFs ERF2 (CA028270) and MYC2 (CA029559), co-expressed in the degenerating pericarp, may be involved, given that they act to integrate JA signaling networks in defense responses (Lorenzo and Solano, 2005). Furthermore, COI1 expression can be identified in the degenerating pericarp. The fact that this gene encodes an F-box protein (BU968442) involved in JA signaling (Xie et al., 1998) underlines further that JA very probably plays an important role in protein degradation in the pericarp.
In addition to genes involved in JA biosynthesis and signaling, an extensive set of ethylene signal transduction pathway genes is activated during pericarp degeneration, including ethylene receptors (ETR3, AL511633), CTR1 (CA029560, BU971644, BU973230, CA024753), a MAP kinase (BU968393), EIN2 (CA026029, CA020555) and ERF2 TF (CA028270; Figure 3b). Genetic analysis of tomato orthologs expressed during fruit ripening suggests that ETR interacts with CTR1, and that signal transduction is effected via a MAP kinase cascade to EIN2. EIN2 is thought to be central to the control of several hormone signal transduction pathways, and can activate the TF ERF (an EREPB family member; Adams-Phillips et al., 2004). The ethylene biosynthesis and signaling pathway depicted in Figure 3(b) is based both on barley gene expression data and on comparative data derived from fruit ripening studies of tomato (Adams-Phillips et al., 2004), a process bearing some similarity to cereal pericarp development.
We suggest that both JA and ethylene are involved in regulating maternal seed tissue PCD, and that the barley ERF2 TF (CA028270) acts as the signal integrator and inducer of PCD-related proteases, as a result of the ERF-binding motifs GCCGCC and CAACA/CACCTG present in their promoters. Whether other EREBP factors are also involved remains unclear. In addition, we note that the two-component response regulator ARR, which acts downstream of ETR in ethylene signaling independently of CTR1 (Hass et al., 2004), is also expressed in degenerating pericarp tissue (CA024680; Table S2). In summary, both hormones may trigger PCD either via the EREBP TFs (Figure 3b) and/or by directly inducing PCD-involved proteases. The latter possibility is suggested by the observation that JA induces a subtilisin-like serine protease in several Arabidopsis tissues (Golldack et al., 2003).