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ABSTRACT Freezing air temperatures kill most leaves, yet the leaves of some species can survive these events. Tracking the temporal and spatial dynamics of freezing remains an impediment to characterizing frost tolerance. Here we deploye time‐lapse imaging and image subtraction analysis, coupled with fine wire thermocouples, to discern the in situ spatial dynamics of freezing and thawing. Our method of analysis of pixel brightness reveals that ice formation in leaves exposed to natural frosts initiates in mesophyll before spreading to veins, and that while ex situ xylem sap freezes near 0°C, in situ xylem sap has a freezing point of −2°C in our model freezing‐resistant species ofLonicera. Photosynthetic rates in leaves that have been exposed to a rapid freeze or thaw do not recover, but leaves exposed to a slow, natural freezing and thawing to −10°C do recover. Using this method, we are able to quantify the spatial formation and timing of freezing events in leaves, and suggest that in situ and ex situ freezing points for xylem sap can differ by more than 4°C depending on the rate of temperature decline. 

Stomatal closure limits transpiration during drought, restricting water potential decline and delaying the onset of embolism. While critical for ensuring survival during drought, the mechanisms driving stomatal closure during drought remain equivocal. The hormone abscisic acid (ABA) will close stomata in seed plants and is synthesized as leaf turgor declines. ABA driven stomatal closure during drought is particularly apparent in species that are more isohydric. In contrast, in species that have a more anisohydric response to drought, like Fagus sylvatica, the importance of ABA in driving stomatal closure during drought is often overlooked or excluded, in place of a hypothesized passive, water potential driven stomatal closure. Here we investigated whether ABA drives stomata closure during a mid-summer drought in field grown F. sylvatica. We found that as leaf water potential declines during a drought, foliage abscisic acid (ABA) levels increase considerably and stomata close. ABA levels in leaves increase as water potentials decline to within 0.3 MPa of turgor loss point, when stomata close. Foliage ABA levels correlate with stomatal conductance throughout a drought and post-drought period. From these results we argue that it is hard to exclude increased ABA levels driving stomatal closure during drought in the anisohydric species F. sylvatica. 

Abstract The phytohormone abscisic acid (ABA) plays a major role in closing the stomata of angiosperms. However, recent reports of some angiosperm species having a peaking-type ABA dynamic, in which under extreme drought ABA levels decline to pre-stressed levels, raises the possibility that passive stomatal closure by leaf water status alone can occur in species from this lineage. To test this hypothesis, we conducted instantaneous rehydration experiments in the peaking-type species Umbellularia californica through a long-term drought, in which ABA levels declined to pre-stress levels, yet stomata remain closed. We found that when ABA levels were lowest during extreme drought, stomata reopen rapidly to maximum rates of gas exchange on instantaneous rehydration, suggesting that the stomata of U. californica were passively closed by leaf water status alone. This contrasts with leaves early in drought, in which ABA levels were highest and stomata did not reopen on instantaneous rehydration. The transition from ABA-driven stomatal closure to passively driven stomatal closure as drought progresses in this species occurs at very low water potentials facilitated by highly embolism-resistant xylem. These results have important implications for understanding stomatal control during drought in angiosperms. 

Abstract Senescence vividly marks the onset of the final stages of the life of a leaf, yet the triggers and drivers of this process are still not fully understood. The hormone abscisic acid (ABA) is an important regulator of leaf senescence in model herbs, but the function of this hormone has not been widely tested in deciduous trees. Here we investigate the importance of ABA as a driver of leaf senescence in winter deciduous trees. In four diverse species we tracked leaf gas exchange, water potential, chlorophyll content, and leaf ABA levels from the end of summer until leaves were abscised or died. We found that no change in ABA levels occurred at the onset of chlorophyll decline or throughout the duration of leaf senescence. To test whether ABA could enhance leaf senescence, we girdled branches to disrupt ABA export in the phloem. Girdling increased leaf ABA levels in two of the species, and this increase triggered an accelerated rate of chlorophyll decline in these species. We conclude that an increase in ABA level may augment leaf senescence in winter deciduous species but that it is not essential for this annual process. 

Abstract The resistance of xylem conduits to embolism is a major factor defining drought tolerance and can set the distributional limits of species across rainfall gradients. Recent work suggests that the proximity of vessels to neighbors increases the vulnerability of a conduit. We therefore investigated whether the relative vessel area of xylem correlates with intra- and inter-generic variation in xylem embolism resistance in species pairs or triplets from the genera Acer, Cinnamomum, Ilex, Quercus and Persea, adapted to environments differing in aridity. We used the optical vulnerability method to assess embolism resistance in stems and conducted anatomical measurements on the xylem in which embolism resistance was quantified. Vessel lumen fraction (VLF) correlated with xylem embolism resistance across and within genera. A low VLF likely increases the resistance to gas movement between conduits, by diffusion or advection, whereas a high VLF enhances gas transport thorough increased conduit-to-conduit connectivity and reduced distances between conduits and therefore the likelihood of embolism propagation. We suggest that the rate of gas movement due to local pressure differences and xylem network connectivity is a central driver of embolism propagation in angiosperm vessels. 

Maintaining water transport in the xylem is critical for vascular plants to grow and survive. The drought-induced accumulation of embolism, when gas enters xylem conduits, causes declines in hydraulic conductance (K) and is ultimately lethal. Several methods can be used to estimate the degree of embolism in xylem, from measuring K in tissues to directly visualising embolism in conduits. One method allowing a direct quantification of embolised xylem area is the optical vulnerability (OV) technique. This method has been used across different organs and has a high spatial and temporal resolution. Here, we review studies using the OV technique, discuss the main advantages and disadvantages of this method, and summarise key advances arising from its use. Vulnerability curves generated by the OV method are regularly comparable to other methods, including the centrifuge and X-ray microtomography. A major advantage of the OV technique over other methods is that it can be simultaneously used to determine in situ embolism formation in leaves, stems and roots, in species spanning the phylogeny of land plants. The OV method has been used to experimentally investigate the spreading of embolism through xylem networks, associate embolism with downstream tissue death, and observe embolism formation in the field. 

Summary Plant specialized 1,4-naphthoquinones present a remarkable case of convergent evolution. Species across multiple discrete orders of vascular plants produce diverse 1,4-naphthoquinones via one of several pathways using different metabolic precursors. Evolution of these pathways was preceded by events of metabolic innovation and many appear to share connections with biosynthesis of photosynthetic or respiratory quinones. Here, we sought to shed light on the metabolic connections linking shikonin biosynthesis with its precursor pathways and on the origins of shikonin metabolic genes. Downregulation of Lithospermum erythrorhizon geranyl diphosphate synthase (LeGPPS), recently shown to have been recruited from a cytoplasmic farnesyl diphosphate synthase (FPPS), resulted in reduced shikonin production and a decrease in expression of mevalonic acid and phenylpropanoid pathway genes. Next, we used LeGPPS and other known shikonin pathway genes to build a coexpression network model for identifying new gene connections to shikonin metabolism. Integrative in silico analyses of network genes revealed candidates for biochemical steps in the shikonin pathway arising from Boraginales-specific gene family expansion. Multiple genes in the shikonin coexpression network were also discovered to have originated from duplication of ubiquinone pathway genes. Taken together, our study provides evidence for transcriptional crosstalk between shikonin biosynthesis and its precursor pathways, identifies several shikonin pathway gene candidates and their evolutionary histories, and establishes additional evolutionary links between shikonin and ubiquinone metabolism. Moreover, we demonstrate that global coexpression analysis using limited transcriptomic data obtained from targeted experiments is effective for identifying gene connections within a defined metabolic network.