Drought effects on the tissue- and cell-specific cytokinin activity in poplar.

Climate change with increasing periods of drought is expected to reduce the yield of biomass crops such as poplars. To combat yield loss, it is important to better understand the molecular mechanisms that control growth under drought. Here, the goal was to resolve the drought-induced changes of active cytokinins, a main growth hormone in plants, at the tissue level in different cell types and organs of poplars (Populus × canescens) in comparison with growth, biomass, leaf shedding, photosynthesis and water potential. Since cytokinin response is mediated by type-A response regulators, ARR5::GUS reporter lines were used to map cytokinin activity histochemically. The expression of PtaRR3 and PtaRR10 was examined in different stem sections. Young leaves showed strong cytokinin activity in the veins and low staining under drought stress, accompanied by diminished leaf expansion. Leaf scars, at positions where drought-shedding occurred, showed strong reduction of cytokinin activity. The pith in the differentiation zone of stem showed high cytokinin activity with distinct, very active parenchymatic cells and enhanced activity close to primary xylem. This pattern was maintained under drought but the cytokinin activity was reduced. Mature phloem parenchymatic cells showed high cytokinin activity and mature wood showed no detectable cytokinin activity. Cytokinin activity in the cambium was apparent as a clear ring, which faded under drought. Xylem-localized cytokinin activities were also mirrored by the relative expression of PtaRR3, whereas PtaRR10 showed developmental but no drought-induced changes. Primary meristems exhibited high cytokinin activity regardless of drought stress, supporting a function of this phytohormone in meristem maintenance, whereas declining cytokinin activities in apical pith tissues and cambium of drought-stressed poplars linked cytokinin in these cell types with the control of primary and secondary growth processes. Changes in cytokinin activity further imply a role in drought avoidance mechanisms of poplars, especially in the reduction of leaf area.

Genes and gene clusters related to genotype and drought-induced variation in saccharification potential, lignin content and wood anatomical traits in Populus nigra.

Wood is a renewable resource that can be employed for the production of second generation biofuels by enzymatic saccharification and subsequent fermentation. Knowledge on how the saccharification potential is affected by genotype-related variation of wood traits and drought is scarce. Here, we used three Populus nigra L. genotypes from habitats differing in water availability to (i) investigate the relationships between wood anatomy, lignin content and saccharification and (ii) identify genes and co-expressed gene clusters related to genotype and drought-induced variation in wood traits and saccharification potential. The three poplar genotypes differed in wood anatomy, lignin content and saccharification potential. Drought resulted in reduced cambial activity, decreased vessel and fiber lumina, and increased the saccharification potential. The saccharification potential was unrelated to lignin content as well as to most wood anatomical traits. RNA sequencing of the developing xylem revealed that 1.5% of the analyzed genes were differentially expressed in response to drought, while 67% differed among the genotypes. Weighted gene correlation network analysis identified modules of co-expressed genes correlated with saccharification potential. These modules were enriched in gene ontology terms related to cell wall polysaccharide biosynthesis and modification and vesicle transport, but not to lignin biosynthesis. Among the most strongly saccharification-correlated genes, those with regulatory functions, especially kinases, were prominent. We further identified transcription factors whose transcript abundances differed among genotypes, and which were co-regulated with genes for biosynthesis and modifications of hemicelluloses and pectin. Overall, our study suggests that the regulation of pectin and hemicellulose metabolism is a promising target for improving wood quality of second generation bioenergy crops. The causal relationship of the identified genes and pathways with saccharification potential needs to be validated in further experiments.

Tissue- and Cell-Specific Cytokinin Activity in Populus × canescens Monitored by ARR5::GUS Reporter Lines in Summer and Winter.

Cytokinins play an important role in vascular development. But knowledge on the cellular localization of this growth hormone in the stem and other organs of woody plants is lacking. The main focus of this study was to investigate the occurrence and cellular localization of active cytokinins in leaves, roots, and along the stem of Populus × canescens and to find out how the pattern is changed between summer and winter. An ARR5::GUS reporter construct was used to monitor distribution of active cytokinins in different tissues of transgenic poplar lines. Three transgenic lines tested under outdoor conditions showed no influence of ARR5::GUS reporter construct on the growth performance compared with the wild-type, but one line lost the reporter activity. ARR5::GUS activity indicated changes in the tissue- and cell type-specific pattern of cytokinin activity during dormancy compared with the growth phase. ARR5::GUS activity, which was present in the root tips in the growing season, disappeared in winter. In the stem apex ground tissue, ARR5::GUS activity was higher in winter than in summer. Immature leaves from tissue-culture grown plants showed inducible ARR5::GUS activity. Leaf primordia in summer showed ARR5::GUS activity, but not the expanded leaves of outdoor plants or leaf primordia in winter. In stem cross sections, the most prominent ARR5::GUS activity was detected in the cortex region and in the rays of bark in summer and in winter. In the cambial zone the ARR5::GUS activity was more pronounced in the dormant than in growth phase. The pith and the ray cells adjacent to the vessels also displayed ARR5::GUS activity. In silico analyses of the tissue-specific expression patterns of the whole PtRR type-A family of poplar showed that PtRR10, the closest ortholog to the Arabidopsis ARR5 gene, was usually the most highly expressed gene in all tissues. In conclusion, gene expression and tissue-localization indicate high activity of cytokinins not only in summer, but also in winter. The presence of the signal in meristematic tissues supports their role in meristem maintenance. The reporter lines will be useful to study the involvement of cytokinins in acclimation of poplar growth to stress.