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Red-heart Chinese fir (Cunninghamia lanceolata) has the advantages of high density and attractive color, making it popular in the market. To date, most studies about stems of woody plants have only been reported at the cytological level because of few living cells. In this study, the xylem was successfully partitioned into three effective sampling areas: sapwood, transition zone, and heartwood. Secondary metabolites, cell survival, and differentially expressed genes in the three sampling areas were, respectively, investigated. First, we identified the phenylpropanoid and flavonoid pathways closely related to color. Based on the chemical structure of secondary metabolites in pathways, two notable directions had been found. Luteolin’s glycosylation products might be the key substances that regulated the color of heartwood in red-heart Chinese fir because of the 1,000-fold difference between red-heart and white-heart. We also found pinocembrin and pinobanksin in Chinese fir, which were rarely reported before. At the cytological level, we believed that the transition zone of red-heart Chinese fir was a critical region for color production because of the fewer living ray parenchyma cells. In addition, transcriptome and quantitative reverse transcription PCR (qRT-PCR) proved that genes regulating the entire phenylpropanoid pathway, upstream of the flavonoid pathway, and some glycosyltransferases were significantly upregulated in the transition zone of red-heart and then colored the heartwood by increasing metabolites. This is the first report on the color-related secondary metabolites regulated by differential genes in red-heart Chinese fir. This study will broaden our knowledge on the effects of metabolites on coloring woody plant xylems.

The AlkB homologs (ALKBH) gene family regulates N6-methyladenosine (m6A) RNA methylation and is involved in plant growth and the abiotic stress response. Poplar is an important model plant for studying perennial woody plants. Poplars typically have a long juvenile period of 7–10 years, requiring long periods of time for studies of flowering or mature wood properties. Consequently, functional studies of the ALKBH genes in Populus species have been limited. Based on AtALKBHs sequence similarity with Arabidopsis thaliana, 23 PagALKBHs were identified in the genome of the poplar 84K hybrid genotype (P. alba  ×  P. tremula var. glandulosa), and gene structures and conserved domains were confirmed between homologs. The PagALKBH proteins were classified into six groups based on conserved sequence compared with human, Arabidopsis, maize, rice, wheat, tomato, barley, and grape. All homologs of PagALKBHs were tissue-specific; most were highly expressed in leaves. ALKBH9B and ALKBH10B are m6A demethylases and overexpression of their homologs PagALKBH9B and PagALKBH10B reduced m6A RNA methylation in transgenic lines. The number of adventitious roots and the biomass accumulation of transgenic lines decreased compared with WT. Therefore, PagALKBH9B and PagALKBH10B mediate m6A RNA demethylation and play a regulatory role in poplar growth and development. Overexpression of PagALKBH9B and PagALKBH10B can reduce the accumulation of H2O2 and oxidative damage by increasing the activities of SOD, POD, and CAT, and enhancing protection for Chl a/b, thereby increasing the salt tolerance of transgenic lines. However, overexpression lines were more sensitive to drought stress due to reduced proline content. This research revealed comprehensive information about the PagALKBH gene family and their roles in growth and development and responsing to salt stress of poplar.

Poplar is not only an important woody plant, but also a model species for molecular plant studies. We identified PagGRF11 (pAxG07Gg0005700), a homolog of the Arabidopsis AtGRF1 (AT4G37740) and AtGRF2 (AT2G22840) gene. We transformed the poplar clone “84K” with PagGRF11, and the transgenic overexpressed plants (PagGRF11-OE) showed plant height reduction (dwarfing), stem diameter increase, internode shortening, and larger leaf area. The Arabidopsis overexpression line grf-oe (Overexpression of PagGRF11 in Arabidopsis), mutant line atgrf (a loss-of-function mutant of the AtGRF1 gene of Arabidopsis thaliana), and mutant trans-complementary line atgrf+oe (overexpression of PagGRF11 in mutant plants (atgrf)) also showed different leaf size phenotypes. Further, tissue sections revealed that increased xylem production was the main cause of stem thickening. Transcriptome differential expression analysis of PagGRF11 overexpressed and control plants showed that PagGRF11 promoted CCCH39(C3H39) expression. The expression profile of CCCH39 in different tissues showed that it was highly expressed in xylem. Yeast single hybrid and instantaneous double luciferase assay results showed that PagGRF11 directly transcribed and activated CCCH39 expression through interaction with cis-acting element GARE (TCTGTTG), thus promoting xylem development. This is the first finding that GRF positively regulates xylem development through CCCH39 expression activation and further suggests that PagGRF11 is a potential target for increasing wood yield.

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