Turning moss into algae

Abstract

Prenylation is a series of lipid posttranslational modifications that are involved in several key aspects of plant development. We recently knocked out every prenylation subunit in Physcomitrella patens. Like in Arabidopsis, knockout of protein farnesyltransferase and protein geranylgeranyltransferase in P. patens does not result in lethality; however, effects on development are extensive. In particular, the knockout of protein geranylgeranyltransferase results in small unicellular plants that resemble algae. Here we perform an analysis of predicted geranylgeranyltransferase target proteins in P. patens, and draw attention to those most likely to play a role in the knockout phenotype.

One central question of developmental biology is: how do individual cells in a multicellular organism acquire their fates, and divide and differentiate accordingly? One answer is that cells constantly signal to each other, conveying positional information, and these signals result in changes in gene expression that drive differentiation and establish cell identity. Signal transduction relies on post-translational modifications that activate or deactivate proteins, promote protein-protein interactions, and determine subcellular localization. Some of the key protein modifications that drive subcellular localization in eukaryotes are prenylation, myristoylation, and acylation, all of which involve the addition of a lipid moiety to specific proteins, promoting their association with membranes or other proteins with hydrophobic domains.^1,2

We are particularly interested in the role of protein prenylation in plant developmental processes. There are three varieties of protein prenylation, including protein farnesylation (the addition of a 15-carbon farnesyl moiety to target proteins), protein geranylgeranylation (the addition of a 20-carbon geranylgeranyl moiety), and protein dual geranylgeranylation (the addition of dual 20-carbon geranylgeranyl moieties, though to date this is only known to target Rab GTPases).^1,3,4 Protein prenyltransferases are heterodimers, consisting of an α and β subunit.⁵ Interestingly, protein farnesyltransferase (PFT) and protein geranylgeranyltransferase (PGGT) share a common α subunit; target and substrate specificity are conferred by the respective β subunits.⁵ Rab geranylgeranyltransferase (RabGGT) consists of distinct α and β subunits.⁵

Studies focusing on mutants in Arabidopsis have elucidated the biological roles of prenylation in plant development.^1,6-12 Elimination of PFT activity (by deletion of the activity of the PFT β subunit, also known as ENHANCED RESPONSE TO ABSCISIC ACID1 or ERA1) results in plants with slightly shorter stature (due to decreased internode length) and slightly slower growth. Most dramatically affected are the shoot and flower meristems: both are wider with more cells, resulting in altered phyllotaxy, a slight increase in floral organ number, and altered floral organ positioning.^6-9 Elimination of PGGT activity in Arabidopsis (by knocking out the PGGT β subunit, also called GERANYLGERANYLTRANSFERASE BETA or GGB) results in plants that resemble wild type under normal growth conditions.¹¹ Elimination of both PFT and PGGT, whether by knocking out their shared α subunit (also known as PLURIPETALA or PLP), or by generating era1 ggb double mutants, result in plants that are very slow growing, with leaf shape defects, severe meristem enlargement, and greatly altered phyllotaxy and floral organ number, especially an increase in petal number.^10,11,13 Still, the plants remain viable and fertile, though fertility is reduced.¹⁰ The only reported mutation in a RabGGT subunit, RAB GERANYLGERANYLTRANSFERASE BETA1 (RGTB1), one of the 2 RabGGT β subunits in Arabidopsis, results in severe growth defects including small, epinastic leaves and extreme loss of apical dominance.¹²

The most surprising aspect of studies of prenylation mutants in Arabidopsis is their relatively mild phenotypes. This is in contrast to PFT and PGGT mutants in animals and fungi, which are generally lethal.^14-20 This is also despite the fact that hundreds of Arabidopsis proteins meet the criteria for being targets of protein prenylation (a C-terminal CaaX amino acid sequence, where C is the prenylated cysteine, a is usually an aliphatic amino acid, and X is usually alanine, cysteine, glutamine, methionine, or serine in the case of PFT, and almost always leucine in the case of PGGT).²¹ We were interested in 2 questions: first, is Arabidopsis an outlier among plants in being able to survive the loss of protein prenylation? And also, are the general developmental roles of prenylation conserved among plants?

To answer these questions we systematically knocked out every protein prenylation subunit in the moss Physcomitrella patens.²² The most striking phenotype is the mutant of the β subunit of PGGT (Ppggb), in which individual cells fail to adhere to each other and fail to differentiate and elongate, resembling unicellular algae, or undifferentiated flowering plant cell culture cells like tobacco BY2 or Arabidopsis suspension cultures. Ppera1 mutants are less severe, as they adhere to each other and establish cell polarity, but do not elongate properly and are delayed in differentiation. Ppplp mutants resemble Ppggb mutants, but the individual cells die at high rates. Thus, PFT and PGGT are not, strictly speaking, required for survival in P. patens, but the phenotypes of loss of PFT and PGGT are comparatively more severe than in Arabidopsis. In addition, there is a definite role in cell fate acquisition and differentiation in Ppggb mutants, similar to the lack of cell differentiation resulting in larger meristems in Arabidopsis plp mutants.

We are currently interested in establishing which target proteins of PGGT are responsible for cell adhesion, polarity establishment, and polar cell elongation. We used a perl script to identify proteins from the sequenced and annotated P. patens genome version 1.6.²³ We identified 61 proteins with a C-terminal CaaL box, compared with 70 in Arabidopsis. We then performed BLAST searches to augment the current annotation and to determine whether the target protein has an Arabidopsis homolog. Interestingly, 14 of the target proteins have no close homolog in Arabidopsis. These target proteins may be responsible for the differences in phenotype between the Ppggb mutants and the Arabidopsis ggb mutants. In addition, we could not assign a function to 18 of the target proteins.

Several of the candidate target proteins may be involved in signal transduction: 4 are protein kinases, one is a phosphatase, and 2 have other roles associated with signal transduction in other systems. These may be significant in cell-cell communication and conferring cell identity.

Two of the putative targets are involved in the endomembrane system, and three additional putative targets may be involved in cell wall synthesis. Both of these categories may affect the integrity of the cell wall, which may contribute to the lack of adhesion in Ppggb mutants.

Another significant class of putative PGGT target proteins is the Rop GTPases, all 4 of which in P. patens have a CaaL box.²⁴ Rop GTPases are involved in polar cell growth in flowering plants, including root hair elongation, pollen tube elongation, and pavement cell lobe elaboration,^25-27 so it is reasonable to speculate that polar cell elongation in P. patens relies on Rop GTPase activity as well.

Seven of the putative target proteins play a role in transcriptional regulation. Plants are unique among eukaryotes in that at least one transcription factor (APETALA1 of Arabidopsis) and select homologs in other species are prenylated, and this prenylation is important for its function.²⁸ It is possible that these prenylated transcription factors also play a role in cell fate determination or other activity.

Further studies of PFT and PGGT target proteins, including the generation of knockout lines of these targets, should lead to fundamental insights into key cell fate determination, cell differentiation, and polar cell elongation processes in P. patens.

Glossary

Abbreviations:

PFT

protein farnesyltransferase

PGGT

protein geranylgeranyltransferase

RabGGT

Rab geranylgeranyltransferase

ERA1

ENHANCED RESPONSE TO ABSCISIC ACID

GGB

GERANYLGERANYLTRANSERASE BETA SUBUNIT

PLP

PLURIPETALA

P. patens

Physcomitrella patens

Ppggb

Physcomitrella patens geranylgeranyltransferase beta subunit mutant

Ppera1

Physcomitrella patens enhanced response to abscisic acid1 mutant

Ppplp

Physcomitrella patens pluripetala mutant

Thole JM, Perroud PF, Quatrano RS, Running MP. Prenylation is required for polar cell elongation, cell adhesion, and differentiation in Physcomitrella patens. Plant J. 2014;78:441–51. doi: 10.1111/tpj.12484.