Advances in Rootstock Breeding of Nut Trees: Objectives and Strategies.

The production and consumption of nuts are increasing in the world due to strong economic returns and the nutritional value of their products. With the increasing role and importance given to nuts (i.e., walnuts, hazelnut, pistachio, pecan, almond) in a balanced and healthy diet and their benefits to human health, breeding of the nuts species has also been stepped up. Most recent fruit breeding programs have focused on scion genetic improvement. However, the use of locally adapted grafted rootstocks also enhanced the productivity and quality of tree fruit crops. Grafting is an ancient horticultural practice used in nut crops to manipulate scion phenotype and productivity and overcome biotic and abiotic stresses. There are complex rootstock breeding objectives and physiological and molecular aspects of rootstock-scion interactions in nut crops. In this review, we provide an overview of these, considering the mechanisms involved in nutrient and water uptake, regulation of phytohormones, and rootstock influences on the scion molecular processes, including long-distance gene silencing and trans-grafting. Understanding the mechanisms resulting from rootstock × scion × environmental interactions will contribute to developing new rootstocks with resilience in the face of climate change, but also of the multitude of diseases and pests.

Uncovering early response to drought by proteomic, physiological and biochemical changes in the almond × peach rootstock 'Garnem'.

Drought affects growth and metabolism in plants. To investigate the changes in root protein function involved in the early response to drought stress, a proteomic analysis in combination to a physiological and biochemical analysis was performed in plants of 'Garnem', an almond × peach hybrid rootstock, subjected to short-term drought stress. Abscisic acid (ABA) accumulation levels increased during the drought exposure, which induced stomatal closure, and thus, minimised water losses. These effects were reflected in stomatal conductance and leaf water potential levels. However, 'Garnem' was able to balance water content and maintain an osmotic adjustment in cell membranes, suggesting a dehydration avoidance strategy. The proteomic analysis revealed significant abundance changes in 29 and 24 spots after 2 and 24 h of drought stress respectively. Out of these, 15 proteins were identified by LC-ESI-MS/MS. The abundance changes of these proteins suggest the influence in drought-responsive mechanisms present in 'Garnem', allowing its adaptation to drought conditions. Overall, our study improves existing knowledge on the root proteomic changes in the early response to drought. This will lead to a better understanding of dehydration avoidance and tolerance strategies, and finally, help in new drought-tolerance breeding approaches.

Identification of water use efficiency related genes in 'Garnem' almond-peach rootstock using time-course transcriptome analysis.

Drought is one of the main abiotic stresses with far-reaching ecological and socioeconomic impacts, especially in perennial food crops such as Prunus. There is an urgent need to identify drought resilient rootstocks that can adapt to changes in water availability. In this study, we tested the hypothesis that PEG-induced water limitation stress will simulate drought conditions and drought-related genes, including transcription factors (TFs), will be differentially expressed in response to this stress. 'Garnem' genotype, an almond × peach hybrid [P. amygdalus Batsch, syn P. dulcis (Mill.) x P. persica (L.) Batsch] was exposed to PEG-6000 solution, and a time-course transcriptome analysis of drought-stressed roots was performed at 0, 2 and 24 h time points post-stress. Transcriptome analysis resulted in the identification of 12,693 unique differentially expressed contigs (DECs) at the 2 h time point, and 7,705 unique DECs at the 24 h time point after initiation of the drought treatment. Interestingly, three drought-induced genes, directly related to water use efficiency (WUE) namely, ERF023 TF; LRR receptor-like serine/threonine-kinase ERECTA; and NF-YB3 TF, were found induced under stress. The RNAseq results were validated with quantitative RT-PCR analysis of eighteen randomly selected differentially expressed contigs (DECs). Pathway analysis in the present study provides valuable information regarding metabolic events that occur during stress-induced signalling in 'Garnem' roots. This information is expected to be useful in understanding the potential mechanisms underlying drought stress responses and drought adaptation strategies in Prunus species.

Preformed and induced mechanisms underlies the differential responses of Prunus rootstock to hypoxia.

Analysis of the transcriptomic changes produced in response to hypoxia in root tissues from two rootstock Prunus genotypes differing in their sensitivity to waterlogging: resistant Myrobalan 'P.2175' (P. cerasifera Erhr.), and sensitive 'Felinem' hybrid [P. amygdalus Batsch × P. persica (L.) Batsch] revealed alterations in both metabolism and regulatory processes. Early hypoxia response in both genotypes is characterized by a molecular program aimed to adapt the cell metabolism to the new conditions. Upon hypoxia conditions, tolerant Myrobalan represses first secondary metabolism gene expression as a strategy to prevent the waste of resources/energy, and by the up-regulation of protein degradation genes probably leading to structural adaptations to long-term response to hypoxia. In response to the same conditions, sensitive 'Felinem' up-regulates a core of signal transduction and transcription factor genes. A combination of PLS-DA and qRT-PCR approaches revealed a set of transcription factors and signalling molecules as differentially regulated in the sensitive and tolerant genotypes including the peach orthologs for oxygen sensors. Apart from providing insights into the molecular processes underlying the differential response to waterlogging of two Prunus rootstocks, our approach reveals a set of candidate genes to be used expression biomarkers for biotech or breeding approaches to waterlogging tolerance.

Physiological and biochemical parameters controlling waterlogging stress tolerance in Prunus before and after drainage.

Waterlogging is associated with poor soil drainage. As a consequence oxygen levels decrease in the root environment inducing root asphyxia and affecting plant growth. Some plants can survive under these conditions triggering complex anatomical and biochemical adaptations, mostly in the roots. Long- and short-term responses to waterlogging stress were compared in two trials using a set of two myrobalans (Prunus cerasifera Erhr), 'P.2175' and 'P.2980', as tolerant rootstocks and two almond × peach [Prunus amygdalus Batsch ×Prunus persica (L.) Batsch] interspecific hybrids, 'Garnem' and 'Felinem', as sensitive ones in two consecutive years. Stomatal conductance and chlorophyll content were measured in the long-term trials to assess survival performance, while the enzyme activities of superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (POD, EC 1.11.1.7), and catalase (CAT, EC 1.11.1.6) were measured in the short-term trials to study early antioxidant response. The incidence of the stress in the root environment was different as a result of the different plant development at the moment of the treatment, as a consequence of different environmental conditions both before and during the treatment between the 2 years. The activity of the different enzymes was higher in the sensitive genotype 'Felinem' than in the tolerant 'P.2175'. This result shows an activation of the antioxidant system and has been observed to depend of the different nature of the roots between the 2 years. As the antioxidant enzymes seem to work more efficiently when roots are more aerated, we cannot conclude that they are responsible for the higher tolerance observed in the myrobalan plums.