The m6A RNA Demethylase ALKBH9B Plays a Critical Role for Vascular Movement of Alfalfa Mosaic Virus in Arabidopsis

The N6-methyladenosine (m6A) pathway has been widely described as a viral regulatory mechanism in animals. We previously reported that the capsid protein (CP) of alfalfa mosaic virus (AMV) interacts with the Arabidopsis m6A demethylase ALKBH9B regulating m6A abundance on viral RNAs (vRNAs) and systemic invasion of floral stems. Here, we analyze the involvement of other ALKBH9 proteins in AMV infection and we carry out a detailed evaluation of the infection restraint observed in alkbh9b mutant plants. Thus, via viral titer quantification experiments and in situ hybridization assays, we define the viral cycle steps that are altered by the absence of the m6A demethylase ALKBH9B in Arabidopsis. We found that ALKBH9A and ALKBH9C do not regulate the AMV cycle, so ALKBH9B activity seems to be highly specific. We also define that not only systemic movement is affected by the absence of the demethylase, but also early stages of viral infection. Moreover, our findings suggest that viral upload into the phloem could be blocked in alkbh9b plants. Overall, our results point to ALKBH9B as a possible new component of phloem transport, at least for AMV, and as a potential target to obtain virus resistance crops.

Radial-axial transport coordination enhances sugar translocation in the phloem vasculature of plants

Understanding the controls of mass transport of photosynthates in the phloem of plants is necessary for describing plant carbon allocation, productivity, and responses to water and thermal stress. While several hypotheses about optimization of phloem structure and function, and limitations of phloem transport under drought have been tested both with models and anatomical data, the true impact of radial water exchange of phloem conduits with their surroundings on mass transport of photosynthates has not been addressed. Here the physics of the Munch mechanism of sugar transport is re-evaluated to include local variations in viscosity resulting from the radial water exchange in two dimensions (axial and radial). Model results show that radial water exchange pushes sucrose away from conduit walls thereby reducing wall frictional stress due to a decrease in sap viscosity and increasing sugar concentration in the central region of the conduit. This leads to increased sugar front speed and axial mass transport for a wide range of phloem conduit lengths and allows sugar transport to operate more efficiently than predicted by previous models. A faster front speed leads to higher phloem resiliency under drought because more sugar can be transported with a smaller pressure gradient.

Sieve tube structural variation in Austrobaileya scandens and its significance for lianescence

Lianas are characterized by large leaf areas and slender stems, a combination of features that require an efficient vascular system. The only extant member of the Austrobaileyaceae, is an endemic twining liana of the tropical Australian forests with well-known xylem hydraulic traits. However, the vascular phloem continuum through aerial organs remains understudied. We analyzed the structure of phloem conduits across leaf veins and stems of A. scandens, combining topological data obtained through light and electron microscopy, with current models of phloem transport. Leaves displayed a low xylem to phloem ratio compared with leaves of other angiosperms, with vascular elements invariant in diameter along the midrib, but tapered across vein hierarchies. Sieve plate pore radii were extremely small: 0.08μm in minor veins, increasing to 0.12μm in the petiole and only to 0.20μm at the base of the stem, tens of meters away. Searcher branches contained tube shaped phloem conduits with a pectin-rich wall, whereas twining stems displayed sieve elements with tangential connections that displayed a greater fraction of the tubes populated with an astonishing number of sieve plates. Hydraulic segmentation of the leaves in Austrobaileyaceae correlate with vesseless leaves that benefit photoassimilate export through volumetric scaling of the sieve tube elements. Yet, compared with canopy dominant trees, the geometrical properties of the sieve tube in twining stems, restrict considerably energy distribution in the sub-canopy layers, potentially favoring the allocation of assimilates toward the elongating branches. Thus, the conductive xylem of twining stems contrasts with a poorly conductive phloem that meets the mechanical constraints of lianescence.

Phloem transport limitation in Huanglongbing affected sweet orange is dependent on phloem-limited bacteria and callose

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is a phloem-limited disease which disrupts citrus production in affected areas. In HLB-affected plants, phloem sieve plate pores accumulate callose, and leaf carbohydrate export is reduced. However, whether HLB causes a reduction in carbohydrate phloem translocation speed, and the quantitative relationships among callose, CLas population, and phloem translocation are still unknown. In this work, a procedure was developed to concurrently measure sugar transport, callose deposition, and relative pathogen population at different locations throughout the stem. Increasing quantities of CLas genetic material were positively correlated with quantity and density of callose deposits, and negatively correlated with phloem translocation speed. Callose deposit quantity was site- and rootstock dependent, and were negatively correlated with phloem translocation speed, suggesting a localized relationship. Remarkably, callose accumulation and phloem translocation disruption in the scion was dependent on rootstock genotype. Regression results suggested that the interaction of Ct values and number of phloem callose depositions, but not their size or density, explained the effects on translocation speed. Sucrose, starch, and sink 14C label allocation data support the interpretation of a transport pathway limitation by CLas infection. This work shows that the interaction of local accumulation of callose and CLas affect phloem transport. Further, the extent of this accumulation is attenuated by the rootstock and provides important information about the disease mechanism of phloem-inhabiting bacteria. Together, these results constitute the first example of a demonstrated transport limitation of phloem function by a microbial infection.

Metaphloem development in the Arabidopsis root tip

The phloem transport network is a major evolutionary innovation that enabled plants to dominate terrestrial ecosystems. In the growth apices, the meristems, apical stem cells continuously produce early, so-called protophloem. This is easily observed in Arabidopsis root meristems, where the differentiation of individual protophloem sieve element precursors into interconnected, conducting sieve tubes is laid out in a spatio-temporal gradient. The mature protophloem eventually collapses as the neighboring metaphloem takes over its function further distal from the stem cell niche. Compared to protophloem, metaphloem ontogenesis is poorly characterized, primarily because its visualization is challenging. Here we describe the improved TetSee protocol to investigate metaphloem development in Arabidopsis root tips in combination with a set of molecular markers. We found that mature metaphloem sieve elements are only observed in the late post-meristematic root although their specification is initiated as soon as protophloem sieve elements enucleate. Moreover, unlike protophloem sieve elements, metaphloem sieve elements only differentiate once they have fully elongated. Finally, our results suggest that metaphloem differentiation is not directly controlled by protophloem-derived cues but rather follows a distinct, robust developmental trajectory.

Triggering Bimodal Radial Stem Growth in Pinus sylvestris at a Drought-Prone Site by Manipulating Stem Carbon Availability

A bimodal radial growth (RG) pattern, i.e., growth peaks in spring and autumn, was repeatedly found in trees in the Mediterranean regions, where summer drought causes reduction or cessation of cambial activity. In a dry inner Alpine valley of the Eastern Alps (Tyrol, Austria, 750 m asl), Pinus sylvestris shows unimodal RG with onset and cessation of cambial activity in early April and late June, respectively. A resumption of cambial activity after intense summer rainfall was not observed in this region. In a field experiment, we tested the hypothesis that early cessation of cambial activity at this drought-prone site is an adaptation to limited water availability leading to an early and irreversible switch of carbon (C) allocation to belowground. To accomplish this, the C status of young P. sylvestris trees was manipulated by physical blockage of phloem transport (girdling) 6 weeks after cessation of cambial cell division. Influence of manipulated C availability on RG was recorded by stem dendrometers, which were mounted above the girdling zone. In response to blockage of phloem flow, resumption of cambial activity was detected above girdling after about 2 weeks. Although the experimentally induced second growth surge lasted for the same period as in spring (c. 2 months), the increment was more than twice as large due to doubling of daily maximum RG rate. After girdling, wood anatomical traits above girdling no longer showed any significant differences between earlywood and latewood tracheids indicating pronounced effects of C availability on cell differentiation. Below girdling, no reactivation of cambial activity occurred, but cell wall thickness of last formed latewood cell was reduced due to lack of C supply after girdling. Intense RG resumption after girdling indicates that cessation of cambial activity can be reversed by manipulating C status of the stem. Hence, our girdling study yielded strong support for the hypothesis that belowground organs exert high C sink strengths on the drought-prone study site. Furthermore, this work highlights the need of in-depth experimental studies in order to understand the interactions between endogenous and exogenous factors on cambial activity and xylem cell differentiation more clearly.

Manipulating phloem transport affects wood formation but not local nonstructural carbon reserves in an evergreen conifer

How variations in carbon supply affect wood formation remains poorly understood in particular in mature forest trees. To elucidate how carbon supply affects carbon allocation and wood formation, we attempted to manipulate carbon supply to the cambial region by phloem girdling and compression during the mid- and late-growing season and measured effects on structural development, CO efflux, and nonstructural carbon reserves in stems of mature white pines. Wood formation and stem CO efflux varied with location relative to treatment (i.e., above or below the restriction). We observed up to twice as many tracheids formed above versus below the treatment after the phloem transport manipulation, whereas cell-wall area decreased only slightly below the treatments, and cell size did not change relative to the control. Nonstructural carbon reserves in the xylem, needles, and roots were largely unaffected by the treatments. Our results suggest that low and high carbon supply affects wood formation, primarily through a strong effect on cell proliferation, and respiration, but local nonstructural carbon concentrations appear to be maintained homeostatically. This contrasts with reports of a decoupling of source activity and wood formation at the whole-tree or ecosystem level, highlighting the need to better understand organ-specific responses, within-tree feedbacks, as well as phenological and ontological effects on sink-source dynamics.