An act of fruitility: investigating the causes of outer arm formation in grapevine (Vitis vinifera L.)

Abstract

A major factor affecting quality in wine grapes is the varying levels of ripeness within a bunch. A significant cause of within bunch variation is the formation of an additional branch at the base of the main bunch, called the outer arm. As part of an industry-driven initiative, this work was tasked with identifying the molecular causes of fruitful outer arm development in grapevine. Two maize mutants shown to affect inflorescence architecture are the ramosa3 and the barren stalk1 genes. The ramosa3 phenotype is due to a mutation in a TREHALOSE PHOSPHATE PHOSPHATSE (TPP) gene, a member of the trehalose biosynthesis pathway. The BARREN STALK1/LAX PANICLE (BA1/LAX1) bHLH transcription factor gene family regulates branching by limiting auxin into pluripotent cells. There were no published studies on these genes in grapevine when this research began. The focus of this work was to identify and characterize the TREHALOSE-6-PHOSPHATE SYNTHASE (TPS), TPP, and BA1/LAX1 gene families. Seven TPS and seven TPP genes were identified in grapevine. Yeast complementation studies showed that only one TPS gene but all TPP genes examined are capable of trehalose biosynthesis. Transcription assays showed varying expression patterns of the VvTPS and VvTPP homologues across different grapevine tissues. Of particular interest was the high expression of some VvTPS and VvTPP genes during inflorescence initiation and differentiation. The VvBA1/LAX1 gene identified in grapevine also showed a range of transcription levels in the tissue types tested, with high expression in tissues undergoing rapid developmental changes. This is in agreement with other studies indicating a role for this transcription factor in auxin signalling. To examine if carbohydrate supply affects fruitful outer arm development in grapevine, physiological and molecular experiments were carried out. Shoots of small or large diameter were harvested from a pruning trial, from which single node cuttings were planted and examined for fruitful outer arm development. Cuttings taken from large diameter shoots had a higher frequency of fruitful outer arms than small diameter shoots for both pruning treatments. Shoots from the severe pruning treatment (pruned to 6 nodes) had a lower frequency of fruitful outer arms. The difference in fruitful outer arm development between the two pruning treatments could not be attributed to diameter size (and carbohydrate status) alone, as both treatments had similar diameter sizes. Transcript screening of several VvTPS and VvTPP genes in four bud developmental stages showed that these genes are expressed similar to several floral pathway genes. This leads us to hypothesize that the level of VvTPS and VvTPP expression, or the metabolites formed during trehalose biosynthesis, may be used as a signal by the plant to indicate carbohydrate status during primordia development to regulate inflorescence architecture. Comparison of buds from the same developmental stage, but collected from the two shoot diameter classes, indicated that diameter may have an effect on transcript levels for many of the VvTPS and VvTPP genes tested. When VvBA1/LAX1 was screened on the same bud tissues, there was a two-fold difference in expression during the budswell developmental stage, indicating that this transcription factor is most active just after dormancy during inflorescence differentiation. There was no significant shoot diameter difference in VvBA1/LAX1 expression, so this gene is not believed to have a role in carbohydrate signalling. The results of this research show that the VvTPS and VvTPP gene families are present and active in grapevine, with a possible role in carbohydrate signalling during important developmental stages. We hypothesize that this signalling is partly responsible for fruitful outer arm development when there is a surplus of carbohydrates in the plant at the time of inflorescence primordia initiation. Furthermore, the expression of VvBA1/LAX1 before budburst indicates that this gene may also play a role in outer arm formation by regulating the differentiation of this organ during the second season of development. Although the results of this research cannot confirm these hypotheses, it has built a foundation to further investigate the role of these gene families in fruitful outer arm development.