This doctoral thesis describes the initial molecular genetic and developmental studies of the soybean (Glycine max L.) gene GmCLAVATA1A (GmCLV1A) illustrating that despite close relatedness to the ‘Autoregulation of Nodulation’ (AON) GmNARK (Nodule Autoregulation Receptor Kinase) gene, its function relates to shoot-organ development and differentiation. The shoot apical meristem (SAM) and its stem cells are responsible for post-embryonic development of all plants. Maintenance of a pluripotent stem cell population and organ initiation are major functions of meristems. In a proposed molecular mechanism derived from the model plant Arabidopsis thaliana, CLAVATA1 (CLV1) plays a central role the CLV-WUSCHEL network to maintain the stem cell population of the SAM. Mutations in AtCLV1 lead to enlarged shoot apices and floral meristems. The soybean genome underwent two rounds of duplication, which caused 75% of the genes to have multiple copies. GmNARK and its duplicate, GmCLV1A, are AtCLV1-like genes and have the highest similarity with AtCLV1 among the leucine rich repeat (LRR) receptor kinases in soybean. GmNARK nodulation controls functions in the leaf and controls the root nodule number through a systemic long distance circuit called ‘Autoregulation of Nodulation (AON)’. Moreover, GmNARK functions locally in the root to regulate nitrate sensing for nodule suppression. GmCLV1A is highly similar to GmNARK with DNA sequence identity in coding areas of 92%. However, little is known about GmCLV1A and the “Arabidopsis” CLAVATA pathway in any legume. This thesis aimed to describe and elucidate the function of GmCLV1A.
GmCLV1A and GmNARK were found to be structurally similar and located in a homeologous genomic environment on soybean chromosomes 11 and 12, respectively. Moreover, a novel, truncated version of GmCLV1A (called GmTrCLV1A) was identified upstream of GmCLV1A, similar to a truncation of the Medicago truncatula autoregulation gene MtSUNN, an orthologue of GmNARK. Like GmNARK, GmCLV1A is expressed in different tissues of the shoot and root at various levels. A recessive loss-of-function allele was isolated for GmCLV1A through TILLING (Targeting Induced Local Lesions IN Genomes). The allele contains a missense mutation (S562L) in a putative S-glycosylation site of the LRR domain of GmCLV1A. The mutant exhibited a plastic and temperature-sensitive phenotype displaying increased basal branching, abnormal leaf development and number, bifurcated pods and veinal leaf development, but normal nodulation. This indicates that GmCLV1A plays a role in regulating the development of different organs but not, like GmNARK, in nodule initiation and growth. This regulating effect is proposed to occur through altered meristem activity and hormonal changes.
To see whether GmCLV1A and GmNARK proteins functionally interact, a Gmclv1a Gmnark double mutant was generated. This Gmclv1a Gmnark mutant exhibited a super-nodulation phenotype with drastic inhibition of shoot growth. This indicates that in addition to the distinct functions of GmCLV1A and GmNARK in separate pathways, they may also share a common function in plant growth and development. In addition, to see how the Gmclv1a mutant’s gene expression changes at shoot and root tip regions and to identify potential components of the CLAVATA-like system in soybean, the shoot and root tip transcriptomes of the Gmclv1a mutant (S562L) and wild type (cv. Forrest) were investigated using RNA-seq. Differential expression in the Gmclv1a mutant was detected mostly for genes involved in transcription, signalling/receptor kinase activity, or defense/stress-response. We identified WUSCHEL-related homeobox 13 as a potential component acting in the CLV system in soybean that is negatively regulated by GmCLV1A and indicative of some commonalities between the Arabidopsis and soybean CLV network. WUS is a homeodomain transcription factor, which is negatively regulated by AtCLV1 in Arabidopsis. Taken together, the findings described here indicate that GmCLV1A acts in shoot architecture with no observable function in root and nodule formation, consistent with the mode of action of AtCLV1 and different from its homeologous paralogue GmNARK. GmCLV1A most likely acts in a gene network similar to the Arabidopsis CLV1 system. Future work using additional mutant material (such as a yet non-existent non-sense mutant of GmCLV1A), further cellular and developmental analyses, and detailed biochemical studies will be needed to evaluate the complex control of stem-cell associated plant development in soybean.