Abstract
BackgroundThe majority of nitrogen accumulating in cereal grains originates from proteins remobilised from vegetative organs. However, interactions between grain filling and remobilisation are poorly understood. We used transcriptome large-scale pyrosequencing of flag leaves, glumes and developing grains to identify cysteine peptidase and N transporter genes playing a role in remobilisation and accumulation of nitrogen in barley.ResultsCombination of already known and newly derived sequence information reduced redundancy, increased contig length and identified new members of cysteine peptidase and N transporter gene families. The dataset for N transporter genes was aligned with N transporter amino acid sequences of rice and Arabidopsis derived from Aramemnon database. 57 AAT, 45 NRT1/PTR and 22 OPT unigenes identified by this approach cluster to defined subgroups in the respective phylogenetic trees, among them 25 AAT, 8 NRT1/PTR and 5 OPT full-length sequences. Besides, 59 unigenes encoding cysteine peptidases were identified and subdivided into different families of the papain cysteine peptidase clade. Expression profiling of full-length AAT genes highlighted amino acid permeases as the group showing highest transcriptional activity. HvAAP2 and HvAAP6 are highly expressed in vegetative organs whereas HvAAP3 is grain-specific. Sequence similarities cluster HvAAP2 and the putative transporter HvAAP6 together with Arabidopsis transporters, which are involved in long-distance transfer of amino acids. HvAAP3 is closely related to AtAAP1 and AtAAP8 playing a role in supplying N to developing seeds. An important role in amino acid re-translocation can be considered for HvLHT1 and HvLHT2 which are specifically expressed in glumes and flag leaves, respectively. PCA and K-means clustering of AAT transcript data revealed coordinate developmental stages in flag leaves, glumes and grains. Phloem-specific metabolic compounds are proposed that might signal high grain demands for N to distantly located plant organs.ConclusionsThe approach identified cysteine peptidases and specific N transporters of the AAT family as obviously relevant for grain filling and thus, grain yield and quality in barley. Up to now, information is based only on transcript data. To make it relevant for application, the role of identified candidates in sink-source communication has to be analysed in more detail.
Highlights
The majority of nitrogen accumulating in cereal grains originates from proteins remobilised from vegetative organs
RNA-seq and sequence assembly mRNA was prepared from barley flag leaves, glumes and caryopses collected at different stages of grain development
Equal amounts of RNA were combined from each stage at 2 day intervals, from 4 days before anthesis up to 24 days after flowering (DAF) for flag leaves and glumes, between anthesis and 24 DAF for caryopses
Summary
The majority of nitrogen accumulating in cereal grains originates from proteins remobilised from vegetative organs. We used transcriptome large-scale pyrosequencing of flag leaves, glumes and developing grains to identify cysteine peptidase and N transporter genes playing a role in remobilisation and accumulation of nitrogen in barley. Flag leaves and glumes maintain their metabolic activity longer than other vegetative tissues, and their contribution to the final grain yield is high [3]. Certain C1A-type (papaintype) cysteine proteases and possibly S10-type serine carboxypeptidases are involved in bulk degradation of stromal proteins during leaf senescence [8]. Both types of proteases are potentially synthesised at the endoplasmatic reticulum and channelled by the secretory pathway, which suggests routing to the lytic vacuolar compartment such as small senescence-associated vacuoles [9]. High expression and strong upregulation of genes encoding papainlike cysteine peptidases suggests an important role for especially those family members in naturally senescing barley leaves between 7 and 21 DAF [7]
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