“What we’ve got here is failure to communicate”

Abstract

ZHOUPI, a unique and highly conserved bHLH transcription factor, controls both endosperm breakdown and embryonic surface formation during Arabidopsis seed development. We have demonstrated that these two processes are distinct, and that ZHOUPI regulates embryonic surface formation via a signaling pathway mediated by the subtilisin-like serine protease ABNORMAL LEAF SHAPE1, and the receptor-kinases GASSHO1 and GASSHO2. Gene expression profiling in mutant backgrounds has permitted the identification of genes whose expression depends on both ZHOUPI and ABNORMAL LEAF SHAPE1 and genes whose expression depends uniquely on ZHOUPI. The latter are presumably involved specifically in endosperm breakdown, and we discuss this poorly understood process in the light of our results. Finally, we consider the potential ancestral role of ZHOUPI and discuss how its relationship with ABNORMAL LEAF SHAPE1 may have evolved.

Keywords: :

• cuticle formation
• embryogenesis
• endosperm breakdown
• seed development
• signaling

The development of angiosperm seed, upon which human life depends, requires the coordinated growth of the embryo and the surrounding endosperm. In Arabidopsis and many other plants the endosperm progressively degrades as it is invaded by the growing embryo during seed development so that at maturity the seed is filled by the embryo and only a single layer of endosperm remains.¹ Surprisingly, little is known about the molecular basis for the breakdown of the endosperm or whether mechanistically it resembles other forms of developmental cell death. Here, mutants in the Arabidopsis ZHOUPI (ZOU) gene may shed light. In zou mutants, the endosperm fails to break down so that at maturity the seed has a small embryo and a pouch of persistent endosperm. The interpretation of this defect is complicated however by a second feature of the mutant phenotype: zou embryos give rise to seedlings with a defective epidermis and cuticle in their cotyledons. This is striking because ZOU is not expressed in the embryo or seedling, but rather in a specialized layer of endosperm (ESR) that surrounds the embryo.² Therefore, ZOU mediates a signal from the ESR to the embryo promoting cuticle and or epidermal development. Unlike wild type embryos which become separated from the surrounding endosperm around the time the endosperm cellularizes, zou embryos remain tightly stuck to the endosperm, presumably due to defects in their cuticles. This raises the problem of whether the persistent endosperm of zou mutants reflects a direct role for ZOU in promoting cell death in ESR cells, or whether it is an indirect effect of the adhesion of embryo to endosperm preventing the embryo from enlarging and invading the endosperm. We recently resolved this problem using a transgenic approach.³

The transcription of ALE1, which encodes an ESR-specific subtilisin-like serine protease⁴ is strongly down regulated in the zou mutant.² Because ale1 mutants have an epidermal phenotype similar to that of zou mutants, ALE1 likely mediates ZOU’s role in promoting embryo cuticle formation. Indeed, when we restored ALE1 expression in zou mutants by introducing an ALE1 transgene controlled by an ESR-specific promoter, the zou epidermal phenotype was ameliorated but the persistent endosperm defect was unchanged.³ This indicates that ZOU positively regulates two distinct processes in seed development: embryo epidermal development through ALE1-mediated signaling and endosperm breakdown through a separate pathway.

ZOU Promotes Signaling from the Endosperm to the Embryo

Our genetic analysis elucidated additional key molecular components of the ALE1-mediated signaling pathway. Backgrounds in which the redundant receptor like kinases GSO1 and GSO2 are lost have a severely defective cotyledon epidermis.⁵ Epistasis between the mutant phenotypes of zou, ale1 and the gso1/gso2 double mutant indicates that they likely function in the same epidermal development pathway.³ ZOU is necessary for transcription of ALE1 in the ESR. Other subtilases have been shown to cleave peptides to give their active forms.^6,7 We propose that this may be the function of ALE1 in the endosperm. A putative peptide, processed by ALE1, could act as a ligand for the embryo-expressed GSO1/GSO2 RLKs; the resulting signal transduction positively regulating embryo epidermal development.

Developmental Cell Death in Plants

Plant cell death is morphologically distinct from the relatively well characterized apoptotic Programmed Cell Death (PCD) of animals and has been broadly categorized into two types, vacuolar and necrotic.⁸ Although many of the key cell death regulators in animals, such as caspases, are not conserved in plants, structurally related proteins, for example metacaspases, may play a role.^9,10 The Hypersensitive Response (HR), which results in localized cell death circumscribing points of infection, is the best studied PCD-like process in plants.¹¹ The HR employs dissociation of the mitochondrial respiratory transport chain to release Reactive Oxygen Species (ROS), which act to disrupt membrane integrity through peroxidation of lipids. Some effectors of HR cell death may also contribute to developmental cell death, but initiation is likely triggered by different mechanisms. Developmental cell death has been characterized in relation to various processes, including organ abscission, integument development, xylem differentiation, senescence and aerenchyma formation.⁸ Although several cell death promoting peptides and corresponding signaling pathways have been identified, in most cases the components are specific for particular processes.^12-14 Endosperm breakdown is in some ways most similar to aerenchyma formation and abscission, in that the cell walls are completely digested but it remains unclear to what extent the underlying mechanisms are similar to these or other developmental cell death pathways. The ZOU gene provides a valuable tool to address this question; because it encodes a bHLH transcription factor, its target genes are likely to include many of the genes initiating or executing endosperm breakdown.

Identifying the Targets of ZOU

We have used RNA sequencing to compare transcriptional profiles of wild-type, ale1 and zou siliques. At the heart stage of embryo development, just before zou seeds become morphologically distinct from wild-type seeds, 180 genes were downregulated more than 2-fold in zou relative to wild type, whereas 85 were upregulated. At the torpedo stage, when the embryo is invading the degrading endosperm, 326 genes were downregulated and 162 were upregulated in zou mutants. The fact that nearly twice as many genes are down as upregulated in mutants suggests that ZOU is a transcriptional activator. When we compared the genes downregulated in ale1 and zou mutants, we found fewer targets overall in ale1 mutants (93 and 189 genes at heart and torpedo, respectively) consistent with the milder ale1 phenotype. A high proportion of the ALE1 targets were also targets of ZOU (60% of genes at heart stage and 46% at torpedo stage). Such an overlap is highly unlikely by chance and reflects the fact that ZOU and ALE1 act in a common pathway. Since ALE1 functions in epidermal development but not endosperm breakdown, the genes that are targets of ZOU but not of ALE1 should be enriched for mediators of the cell-death promoting role of ZOU. We identified a total of 336 ZOU specific targets at heart and/or torpedo stage. Genes encoding key regulators of developmental cell death, including peptide ligands such as INFLORESCENCE DEFICIENT IN ABSCISSION¹² or KISS OF DEATH,¹³ are not regulated by ZOU, which suggests that endosperm breakdown may involve other factors. Gene Ontology analysis shows an over-representation of cell death-associated ontologies, such as “ROS Responsive” and “Cell Wall Modification.” The prevalence of cell wall modification genes is consistent with the fact that cell walls are completely degraded during endosperm breakdown, while the ROS responsive category may reflect overlap with downstream components of the HR response. Mutant and gain of function analyses may help to test the role of these candidate genes in cell death.

Ancestral Roles of ZOU

Phylogenetic analysis indicates that ZOU is ancient.^2,3 It is conserved not only in angiosperms but also in gymnosperms and the lycophyte Selaginella moellendorfii. Intriguingly the ZOU target ALE1 does not have clear orthologs outside angiosperms. It is possible therefore that the function of ZOU in tissue breakdown may be ancient while its role in controlling ALE1 to promote epidermal development may have been acquired during the angiosperm radiation. Linking female gametophyte proliferation to fertilization (the advent of endosperm) may have presented new challenges in epidermal development for angiosperm embryos. The concomitant development of the endosperm with the embryo, and in particular its cellularization, may have made it difficult for the embryo to maintain a clear boundary in the form of a cuticularized epidermal cell wall. Consistent with this, ZOU is not required for cuticle formation in the absence of endosperm, for example in adult leaves and flowers, while the phenomenon of somatic embryogenesis indicates that endosperm may not be needed for cuticle development. Within the angiosperms, ZOU is conserved in cereals such as rice and maize which are genetically accessible. However, endosperm development in cereals is different from that in Arabidopsis as the bulk of the endosperm persists at seed maturity and is not degraded until after germination. Determining where ZOU is expressed in cereals and whether mutation affects endosperm and/or embryonic development will therefore be interesting. ZOU is also conserved in dicot species whose seed have relatively persistent endosperm, such as castor bean and grape, in which it will be possible to test the relative duration of ZOU expression during seed development.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.