XND1 regulates secondary wall deposition in xylem vessels through inhibition of VND functions

Secondary wall deposition in xylem vessels is activated by Vascular-Related NAC Domain proteins (VNDs) that belong to a group of secondary wall NAC (SWN) transcription factors. In contrast, Xylem NAC Domain1 (XND1) negatively regulates secondary wall deposition in xylem vessels when overexpressed. The mechanism by which XND1 exerts its functions remains elusive. We employed the promoter of the fiber-specific Secondary Wall-Associated NAC Domain1 (SND1) gene to ectopically express XND1 in fiber cells to investigate its mechanism of action on secondary wall deposition. Ectopic expression of XND1 in fiber cells severely diminished their secondary wall deposition and drastically reduced the expression of SWN-regulated downstream transcription factors and secondary wall biosynthetic genes but not that of the SWN genes themselves. Transactivation analyses revealed that XND1 specifically inhibited SWN-activated expression of these downstream genes but not their MYB46-activated expression. Both the NAC domain and the C-terminus of XND1 were required for its inhibitory function and its NAC domain interacted with the DNA-binding domains of SWNs. XND1 was shown to be localized in the cytoplasm and the nucleus and its co-expression with VND6 resulted in cytoplasmic sequestration of VND6. Furthermore, the C-terminus of XND1 was indispensable for the XND1-mediated cytoplasmic retention of VND6 and its fusion to VND6 was able to direct VND6 to the cytoplasm and render it unable to activate gene expression. Since the XND1 gene is specifically expressed in xylem cells, these results indicate that XND1 acts through inhibiting VND functions to negatively regulate secondary wall deposition in xylem vessels.

Transcriptomics and Proteomics Reveal the Cellulose and Pectin Metabolic Processes in the Tension Wood (Non-G-Layer) of Catalpa bungei

Catalpa bungei is an economically important tree with high-quality wood and highly valuable to the study of wood formation. In this work, the xylem microstructure of C. bungei tension wood (TW) was observed, and we performed transcriptomics, proteomics and Raman spectroscopy of TW, opposite wood (OW) and normal wood (NW). The results showed that there was no obvious gelatinous layer (G-layer) in the TW of C. bungei and that the secondary wall deposition in the TW was reduced compared with that in the OW and NW. We found that most of the differentially expressed mRNAs and proteins were involved in carbohydrate polysaccharide synthesis. Raman spectroscopy results indicated that the cellulose and pectin content and pectin methylation in the TW were lower than those in the OW and NW, and many genes and proteins involved in the metabolic pathways of cellulose and pectin, such as galacturonosyltransferase (GAUT), polygalacturonase (PG), endoglucanase (CLE) and β-glucosidase (BGLU) genes, were significantly upregulated in TW. In addition, we found that the MYB2 transcription factor may regulate the pectin degradation genes PG1 and PG3, and ARF, ERF, SBP and MYB1 may be the key transcription factors regulating the synthesis and decomposition of cellulose. In contrast to previous studies on TW with a G-layer, our results revealed a change in metabolism in TW without a G-layer, and we inferred that the change in the pectin type, esterification and cellulose characteristics in the TW of C. bungei may contribute to high tensile stress. These results will enrich the understanding of the mechanism of TW formation.

Analysis of Orthologous SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) Promotor Activity in Herbaceous and Woody Angiosperms

SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) is a master regulator of fibre secondary wall deposition in Arabidopsis thaliana (Arabidopsis), with homologs in other angiosperms and gymnosperms. However, it is poorly understood to what extent the fibre-specific regulation of the SND1 promoter, and that of its orthologs, is conserved between diverged herbaceous and woody lineages. We performed a reciprocal reporter gene analysis of orthologous SND1 promoters from Arabidopsis (AthSND1), Eucalyptus grandis (EgrNAC61) and Populus alba × P. grandidentata (PagWND1A) relative to secondary cell wall-specific Cellulose Synthase4 (CesA4) and CesA7 promoters, in both a non-woody (Arabidopsis) and a woody (poplar) system. β-glucuronidase (GUS) reporter analysis in Arabidopsis showed that the SND1 promoter was active in vascular tissues as previously reported and showed interfascicular and xylary fibre-specific expression in inflorescence stems, while reporter constructs of the woody plant-derived promoters were partial to the (pro)cambium-phloem and protoxylem. In transgenic P. tremula × P. alba plants, all three orthologous SND1 promoters expressed the GUS reporter similarly and preferentially in developing secondary xylem, ray parenchyma and cork cambium. Ours is the first study to reciprocally test orthologous SND1 promoter specificity in herbaceous and woody species, revealing diverged regulatory functions in the herbaceous system.

Is size an issue of time? Relationship between the duration of xylem development and cell traits

Background and Aims Secondary growth is a process related to the formation of new cells that increase in size and wall thickness during xylogenesis. Temporal dynamics of wood formation influence cell traits, in turn affecting cell patterns across the tree ring. We verified the hypothesis that cell diameter and cell wall thickness are positively correlated with the duration of their differentiation phases. Methods Histological sections were produced by microcores to assess the periods of cell differentiation in black spruce [Picea mariana (Mill.) B.S.P.]. Samples were collected weekly between 2002 and 2016 from a total of 50 trees in five sites along a latitudinal gradient in Quebec (Canada). The intra-annual temporal dynamics of cell differentiation were estimated at a daily scale, and the relationships between cell traits and duration of differentiation were fitted using a modified von Bertalanffy growth equation. Key Results At all sites, larger cell diameters and cell wall thicknesses were observed in cells that experienced a longer period of differentiation. The relationship was a non-linear, decreasing trend that occasionally resulted in a clear asymptote. Overall, secondary wall deposition lasted longer than cell enlargement. Earlywood cells underwent an enlargement phase that lasted for 12 d on average, while secondary wall thickness lasted 15 d. Enlargement in latewood cells averaged 7 d and secondary wall deposition occurred over an average of 27 d. Conclusions Cell size across the tree ring is closely connected to the temporal dynamics of cell formation. Similar relationships were observed among the five study sites, indicating shared xylem formation dynamics across the entire latitudinal distribution of the species.The duration of cell differentiation is a key factor involved in cell growth and wall thickening of xylem, thereby determining the spatial variation of cell traits across the tree ring.

Apical control of xylem formation in the pine stem. I. Auxin effects and distribution of assimilates

The effect of IAA upon cambial activity, xylem differentiation and translocation of assimilates from the lateral shoot was investigated in spring and late summer in decapitated and ring-barked young trees of <em>Pinus silvestris</em> in the forest stand. Decapitation interrupted cambial xylem production in the uppermost part of the main stem of decapitated trees in spring and late summer, regardless of whether lateral branches below were growing, dormant or disbudded, and the contact through phloem with the roots was maintained or severed. Auxin supplied to the decapitated stems caused an increasing stimulation of cambial xylem production in spring. It also stimulated cambial activity in August but was ineffective in September. Apical control of cambial xylem production was strongly dependent upon the continuity of phloem and/or cambial tissues of the decapitated main-stem-section with lower parts of the plant. Decapitation of the stem strongly reduced the daily rate of cell wall deposition in the cambial xylem derivatives which on the day the experiment started constituted the zones of radial enlargement and maturation. This reduction limited progressively secondary wall deposition in consecutive maturing tracheids even though the cells differentiated longer. Irrespective of the season, auxin prevented the effect of decapitation in cells which were already differentiating when the experiment started as well as extension of the maturation phase. The effect of auxin was somewhat reduced when the lateral branches were additionally decapitated in early summer. In early summer auxin caused a significant increase of the daily rate of cell wall deposition in cells of the cambial zone or the newly produced ones, thus resulting in formation of progressively thicker secondary walls. Late in summer assimilates were transported mostly to the lower part of the stem. Decapitation changed the intact tree pattern of assimilate distribution, increasing the transport in spring and reducing it later in the summer. Prevention of the contact with roots via phloem and cambium in spring (by ring-barking the stem at tree base) decreased decapitation-induced downward transport of assimilates. Application of auxin to the decapitated uppermost segment of the main stem resulted in a significant increase of assimilate translocation into the stem. At least two mechanisms of auxin involvement in regulation of the rate of secondary wall deposition in pine stem tracheids can be considered: (a) induction (or activation) of the cell wall metabolic potential which seems to occur during meristematic or early radial enlargement phases of tracheid differentiation, and (b) regulation of substrate availability during the phase of tracheid maturation.