Human utilization of the mulberry–silkworm interaction started at least 5,000 years ago and greatly influenced world history through the Silk Road. Complementing the silkworm genome sequence, here we describe the genome of a mulberry species Morus notabilis. In the 330-Mb genome assembly, we identify 128 Mb of repetitive sequences and 29,338 genes, 60.8% of which are supported by transcriptome sequencing. Mulberry gene sequences appear to evolve ~3 times faster than other Rosales, perhaps facilitating the species’ spread worldwide. The mulberry tree is among a few eudicots but several Rosales that have not preserved genome duplications in more than 100 million years; however, a neopolyploid series found in the mulberry tree and several others suggest that new duplications may confer benefits. Five predicted mulberry miRNAs are found in the haemolymph and silk glands of the silkworm, suggesting interactions at molecular levels in the plant–herbivore relationship. The identification and analyses of mulberry genes involved in diversifying selection, resistance and protease inhibitor expressed in the laticifers will accelerate the improvement of mulberry plants.

Aquaporins are water channel proteins that facilitate the passage of water through biological membranes and play a crucial role in plant growth. We show that ethylene treatment significantly reduced petal size, inhibited expansion of petal abaxial subepidermal cells, and decreased petal water content in rose (Rosa hybrida 'Samantha'). Here, we report the isolation of a plasma membrane aquaporin (PIP) gene, Rh-PIP2;1, and characterized its potential role in ethylene-inhibited petal expansion. Rh-PIP2;1 is mainly localized on the plasma membrane and belongs to the class 2 subfamily of PIP proteins. We show that Rh-PIP2;1 is an active water channel. The transcripts of Rh-PIP2;1 are highly abundant in petal epidermal cells, especially in the abaxial subepidermal cells. The expression of Rh-PIP2;1 is highly correlated with petal expansion and tightly down-regulated by ethylene. Furthermore, we demonstrate that in Rh-PIP2;1-silenced flowers, petal expansion was greatly inhibited and anatomical features of the petals were similar to those of ethylene-treated flowers. We argue that Rh-PIP2;1 plays an important role in petal cell expansion and that ethylene inhibits petal expansion of roses at least partially by suppressing Rh-PIP2;1 expression.

Dehydration inhibits petal expansion resulting in abnormal flower opening and results in quality loss during the marketing of cut flowers. We constructed a suppression subtractive hybridization library from rose (Rosa hybrida) flowers containing 3,513 unique expressed sequence tags and analyzed their expression profiles during cycles of dehydration. We found that 54 genes were up-regulated by the first dehydration, restored or even down-regulated by rehydration, and once again up-regulated by the second dehydration. Among them, we identified a putative NAC family transcription factor (RhNAC2). With transactivation activity of its carboxyl-terminal domain in yeast (Saccharomyces cerevisiae) cell and Arabidopsis (Arabidopsis thaliana) protoplast, RhNAC2 belongs to the NAC transcription factor clade related to plant development in Arabidopsis. A putative expansin gene named RhEXPA4 was also dramatically up-regulated by dehydration. Silencing RhNAC2 or RhEXPA4 in rose petals by virusinduced gene silencing significantly decreased the recovery of intact petals and petal discs during rehydration. Overexpression of RhNAC2 or RhEXPA4 in Arabidopsis conferred strong drought tolerance in the transgenic plants. RhEXPA4 expression was repressed in RhNAC2-silenced rose petals, and the amino-terminal binding domain of RhNAC2 bound to the RhEXPA4 promoter. Twenty cell wall-related genes, including seven expansin family members, were up-regulated in Arabidopsis plants overexpressing RhNAC2. These data indicate that RhNAC2 and RhEXPA4 are involved in the regulation of dehydration tolerance during the expansion of rose petals and that RhEXPA4 expression may be regulated by RhNAC2.

Drought and high salinity are major environmental conditions limiting plant growth and development. Expansin is a cell-wall-loosening protein known to disrupt hydrogen bonds between xyloglucan and cellulose microfibrils. The expression of expansin increases in plants under various abiotic stresses, and plays an important role in adaptation to these stresses. We aimed to investigate the role of the RhEXPA4, a rose expansin gene, in response to abiotic stresses through its overexpression analysis in Arabidopsis. In transgenic Arabidopsis harboring the Pro RhEXPA4 ::GUS construct, RhEXPA4 promoter activity was induced by abscisic acid (ABA), drought and salt, particularly in zones of active growth. Transgenic lines with higher RhEXPA4 level developed compact phenotypes with shorter stems, curly leaves and compact inflorescences, while the lines with relatively lower RhEXPA4 expression showed normal phenotypes, similar to the wild type (WT). The germination percentage of transgenic Arabidopsis seeds was higher than that of WT seeds under salt stress and ABA treatments. Transgenic plants showed enhanced tolerance to drought and salt stresses: they displayed higher survival rates after drought, and exhibited more lateral roots and higher content of leaf chlorophyll a under salt stress. Moreover, high-level RhEXPA4 overexpressors have multiple modifications in leaf blade epidermal structure, such as smaller, compact cells, fewer stomata and midvein vascular patterning in leaves, which provides them with more tolerance to abiotic stresses compared to mild overexpressors and the WT. Collectively, our results suggest that RhEXPA4, a cell-wall-loosening protein, confers tolerance to abiotic stresses through modifying cell expansion and plant development in Arabidopsis.

SummaryPetal cell expansion depends on cell wall metabolism, changes in cell turgor pressure and restructuring of the cytoskeleton, and recovery ability of petal cell expansion is defined as an indicator of dehydration tolerance in flowers. We previously reported that RhNAC2, a development-related NAC domain transcription factor, confers dehydration tolerance through regulating cell wall-related genes in rose petals. Here, we identify RhNAC3, a novel rose SNAC gene, and its expression in petals induced by dehydration, wounding, exogenous ethylene and abscisic acid (ABA). Expression studies in Arabidopsis protoplasts and yeast show that RhNAC3 has transactivation activity along its full length and in the carboxyl-terminal domain. Silencing RhNAC3 in rose petals by virus-induced gene silencing (VIGS) significantly decreased the cell expansion of rose petals under rehydration conditions. In total, 24 of 27 osmotic stress-related genes were down-regulated in RhNAC3-silenced rose petals, while only 4 of 22 cell expansion-related genes were down-regulated. Overexpression of RhNAC3 in Arabidopsis gave improved drought tolerance, with lower water loss of leaves in transgenic plants. Arabidopsis ATH1 microarray analysis showed that RhNAC3 regulated the expression of stressresponsive genes in overexpressing lines, and further analysis revealed that most of the RhNAC3-up-regulated genes were involved in the response to osmotic stress. Comparative analysis revealed that different transcription regulation existed between RhNAC3 and RhNAC2. Taken together, these data indicate that RhNAC3, as a positive regulator, confers dehydration tolerance of rose petals mainly through regulating osmotic adjustment-associated genes.