Conserved and unique features of the homeologous maize Aux/IAA proteins ROOTLESS WITH UNDETECTABLE MERISTEM 1 and RUM1-like 1.

The maize (Zea mays L.) Aux/IAA protein RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEM 1) is a key regulator of lateral and seminal root formation. An ancient maize genome duplication resulted in the emergence of its homeolog rum1-like1 (rul1), which displays 92% amino acid sequence identity with RUM1. Both, RUL1 and RUM1 exhibit the canonical four domain structure of Aux/IAA proteins. Moreover, both are localized to the nucleus, are instable and have similar short half-lives of ~23min. Moreover, RUL1 and RUM1 can be stabilized by specific mutations in the five amino acid degron sequence of domain II. In addition, proteins encoded by both genes interact in vivo with auxin response factors (ARFs) such as ZmARF25 and ZmARF34 in protoplasts. Although it was demonstrated that RUL1 and RUM1 can homo and heterodimerize in vivo, rul1 expression is independent of rum1. Moreover, on average rul1 expression is ~84-fold higher than rum1 in the 12 tested tissues and developmental stages, although the relative expression levels in different root tissues are very similar. While RUM1 and RUL1 display conserved biochemical properties, yeast-two-hybrid in combination with BiFC experiments identified a RUM1-associated protein 1 (RAP1) that specifically interacts with RUM1 but not with RUL1. This suggests that RUM1 and RUL1 are at least in part interwoven into different molecular networks.

LATERAL ROOT PRIMORDIA 1 of maize acts as a transcriptional activator in auxin signalling downstream of the Aux/IAA gene rootless with undetectable meristem 1.

Only little is known about target genes of auxin signalling downstream of the Aux/IAA-ARF module. In the present study, it has been demonstrated that maize lateral root primordia 1 (lrp1) encodes a transcriptional activator that is directly regulated by the Aux/IAA protein ROOTLESS WITH UNDETECTABLE MERISTEM 1 (RUM1). Expression of lrp1 is confined to early root primordia and meristems and is auxin-inducible. Based on its primary protein structure, LRP1 is predicted to be a transcription factor. This notion is supported by exclusive LRP1 localization in the nucleus and its ability to activate downstream gene activity. Based on the observation that lrp1 transcription is completely repressed in the semi-dominant gain of function mutant rum1, it was demonstrated that the lrp1 promoter is a direct target of RUM1 proteins. Subsequently, promoter activation assays indicated that RUM1 represses the expression of a GFP reporter fused to the native promoter of lrp1. Constitutive repression of lrp1 in rum1 mutants is a consequence of the stability of mutated rum1 proteins which cannot be degraded by the proteasome and thus constitutively bind to the lrp1 promoter and repress transcription. Taken together, the repression of the transcriptional activator lrp1 by direct binding of RUM1 to its promoter, together with specific expression of lrp1 in root meristems, suggests a function in maize root development via the RUM1-dependent auxin signalling pathway.

Rootless with undetectable meristem 1 encodes a monocot-specific AUX/IAA protein that controls embryonic seminal and post-embryonic lateral root initiation in maize.

The maize (Zea mays L.) rum1-R (rootless with undetectable meristems 1-Reference) mutant does not initiate embryonic seminal roots and post-embryonic lateral roots at the primary root. Map-based cloning revealed that Rum1 encodes a 269 amino acid (aa) monocot-specific Aux/IAA protein. The rum1-R protein lacks 26 amino acids including the GWPPV degron sequence in domain II and part of the bipartite NLS (nuclear localization sequence). Significantly reduced lateral root density (approximately 35%) in heterozygous plants suggests that the rum1-R is a semi-dominant mutant. Overexpression of rum1-R under the control of the maize MSY (Methionine SYnthase) promoter supports this notion by displaying a reduced number of lateral roots (31-37%). Functional characterization suggests that Rum1 is auxin-inducible and encodes a protein that localizes to the nucleus. Moreover, RUM1 is unstable with a half life time of approximately 22 min while the mutant rum1-R protein is very stable. In vitro and in vivo experiments demonstrated an interaction of RUM1 with ZmARF25 and ZmARF34 (Z. mays AUXIN RESPONSE FACTOR 25 and 34). In summary, the presented data suggest that Rum1 encodes a canonical Aux/IAA protein that is required for the initiation of embryonic seminal and post-embryonic lateral root initiation in primary roots of maize.

Regulation of the pericycle proteome in maize (Zea mays L.) primary roots by RUM1 which is required for lateral root initiation.

Lateral roots are all roots that are initiated in the pericycle cell layer of other roots during postembryonic development. The maize (Zea mays L.) mutant rum1 (rootless with undetectable meristems 1) does not initiate lateral roots in the primary root. In the present study, two-dimensional electrophoresis proteome profiles of three biological replicates of pericycle cells isolated from the differentiation zone of 2.5-day-old wild-type and rum1 primary roots were generated. This early developmental stage was selected in order to analyze histologically similar cells before the initiation of lateral roots in wild-type primary roots. In total, 418 proteins were reproducibly detected on all six gels after fluorescent staining with Flamingo dye. Among those, twelve proteins were differentially accumulated between wild-type and rum1 pericycle cells (Fc > 2; p < 0.05). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) identified eight of the twelve proteins. Six proteins were related to metabolism, one protein belonged to the class of disease and defense, and one protein was related to development. Six of the eight proteins have not been previously localized to the pericycle. Moreover, the slight overlap between proteins and transcripts that are differentially accumulated in the maize pericycle between wild-type and rum1 underscores the importance of posttranscriptional protein modifications that cannot be detected on the RNA level. The differential accumulation of proteins in rum1 and wild-type pericycle cells of the primary root suggests that the abundance of these proteins could be regulated by RUM1.