Cotyledon explants of walnut (Juglans regia) have been shown to generate adventitious roots on growth regulator-free medium. The spatial distribution of endogenous indole-3-acetic acid (IAA) and its dynamic changes during adventitious root formation were investigated using an in situ immunohistochemical approach. Before root induction, IAA signal was distributed throughout the freshly excised cotyledon explants. During provascular bundle differentiation, the IAA signal was mainly located in the provascular bundles. At the stage of annular meristematic zones formation, the IAA signal was mainly distributed in the meristematic zones and decreased in the vascular bundles and cotyledonous parenchyma. As primordia formed, the IAA signal became localized in the root primordia and gradually disappeared in the meristematic zones. In emerging roots, the IAA signal was mainly localized in the root cap and root meristem. These results suggest that accumulation of IAA in the provascular bundles may induce vascular differentiation and the increase in IAA through meristematic zones may be responsible for the adventitious root formation from walnut cotyledons. The direct evidence presented here indicates that IAA accumulated in the meristematic zones is not the sole signal needed to induce adventitious root.
We used anti-indole-3-acetic acid (IAA) monoclonal antibodies to monitor the temporal and spatial pattern of IAA during pistillate flower bud differentiation in the walnut (Juglans regia) cultivar Liaoning 1. Based on morphological changes, the process of pistillate flower bud differentiation was divided into five stages. The flower induction stage, which includes the early phase, midphase, and late phase, persisted from 25 Apr. to the end of May. The pedicel differentiation stage began on 5 June. The bract primordium stage began on 25 June and persisted through mid-March of the next year. Both the perianth and pistil differentiation stages persisted for nearly 2 weeks. During the floral induction period, little IAA was present in the shoot apical meristem (SAM); hence, the SAM may not always be a site of IAA production. IAA was obviously concentrated in cells of the first several layers of the SAM during pedicel primordium formation. High levels of IAA were also noted in the phyllome, young leaf tips, and vascular bundle of leaves and gemmae. This direct evidence indicates that no close relationship exists between IAA and physiological differentiation; instead, IAA may strongly affect morphogenesis. These findings comprise a first step toward elucidating the walnut flowering mechanism.
The roles of abscisic acid (ABA) and nitric oxide (NO) and the relationship between NO and ABA on chilling resistance and activation of antioxidant activities in walnut (Juglans regia) shoots in vitro under chilling stress were investigated. Walnut shoots were treated with ABA, the NO donor sodium nitroprusside (SNP), ABA in combination with the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO), PTIO, SNP in combination with the ABA biosynthesis inhibitor fluridone (Flu), and Flu. Their effects on chilling tolerance, reactive oxygen species (ROS) levels, and the antioxidant defense system were analyzed. The results showed that ABA treatment markedly alleviated the decreases in the maximal photochemical efficiency and survival and the increases in electrolyte leakage and lipid peroxidation induced by chilling stress, suggesting that application of ABA could improve the chilling tolerance. Further analyses showed that ABA enhanced antioxidant defense and slowed down the accumulation of ROS caused by chilling. Similar results were observed when exogenous SNP was applied. ABA in combination with PTIO or PTIO alone differentially abolished these protective effects of ABA. However, treatment with NO in combination with Flu or Flu alone did not affect the SNP-induced protective effect against CI or the activation of antioxidant activities under conditions of chilling stress. In addition, ABA treatment increased the NO content under chilling conditions, which was suppressed by the ABA biosynthesis inhibitor Flu or NO scavenger PTIO. Conversely, SNP application induced the same ABA rise observed in control plants in response to chilling. Taken together, these results suggested that ABA may confer chilling tolerance in walnut shoots in vitro by enhancing the antioxidant defense system, which is partially mediated by NO, preventing the overproduction of ROS to alleviate the oxidative injury induced by chilling.